Effects of serum, tissue extract, conditioned medium, and culture substrata on neurite appearance from spinal cord explants of chick embryo

Developmental Brain Research, 4 (1982) 303-312 Elsevier Biomedical Press

303

Effects of Serum, Tissue Extract, Conditioned Medium, and Culture Substrata on Neurite Appearance from Spinal Cord Explants of Chick Embryo HIDEAKI TANAKA, MICHIZO SAKAI and KUNIHIKO OBATA Department of Pharmacology, Gunma University School of Medicine, Maebashi 371 (Japan) (Accepted December 3rd, 1981) Key words: primary culture - - chick embryo - - spinal cord explant - - neurite appearance - conditioned medium - - culture substratum

The effects of serum, tissue extracts, conditioned medium (CM), and culture substrata on neurite appearance from spinal cord explants of 6- to 8-day-old chick embryos were investigated. In Eagle's minimum essential medium (MEM) with no supplement neurites from explants did not appear on collagen coating but on polyornithine coating (PORN). It is concluded that cell-to-substratum interaction is important in neurite appearance. CM, serum and tissue extract potentiated neurite appearance, but their activities were highly dependent on the coating. The amount of collagen was also crucial. On collagen, neurite apperance was observed only when promoting substances were present. CM and serum contained at least two components; one affected neurite appearance after deposition on collagen and the other affected neurite appearance when present in the culture medium. The former was included also in tissue extracts. Both of adsorbable and non-adsorbable components from any origin were necessary for effective induction of neurite appearance. Heat treatment and dialysis differentiated these active components. On PORN, CM highly potentiated neurite appearance. The activity of the CM was reproduced by its low molecular weight fraction. Serum also promoted neurite appearance, but to a lesser extent than CM. The effect of tissue extract was not remarkable. INTRODUCTION Some peripheral neurons are k n o w n to require specific substances for survival and development b o t h in vivo and in vitro. Nerve growth factor ( N G F ) is essential to the survival and maturation o f sympathetic and some sensory neurons (refs. 15 and 26 for reviews). Chick ciliary ganglion cells have been reported to require cholinergic neuronotrophic factors for survival in vitro 1,1z,19,28. Their cell death, which reduces the n u m b e r o f ganglion cells by half during embryonic development, is suggested to be related to their innervating target muscles in vivo 13. These findings support the hypothesis that peripheral efferent neurons require trophic mediators from their innervating target tissue or peripheral glia (satellite and Schwann cells) for survival and maturation 27. However, little is k n o w n about trophic relationships in the central nervous system. Spinal cord cells can be cultured and maintained in medi0165-3806/82/0000-0000/$02.75 © Elsevier Biomedical Press

u m supplemented with serum or chick embryo extract s with no specific g r o w t h stimulating factors. This suggests that spinal cord cells do not require exogenous specific trophic factors and/or that serum and e m b r y o extract contain trophic factors. Facilitation o f neurite growth from sensory or sympathetic ganglion cells following transplantation o f mouse sarcoma tissue into chick embryos was first observed in vivo. However, the active factor ( N G F ) responsible for the facilitation o f neurite growth was purified and characterized in vitro, using neurite outgrowth f r o m sensory ganglion explants as an indicator o f its activity15, z6. Observation o f neurite outgrowth in vitro is a useful bioassay system for N G F . Conditioned medium ~CM) o f skeletal and heart muscles highly potentiated neurite growth f r o m spinal cord explants and isolated cells7,11, 25. This suggested that a g r o w t h - p r o m o t i n g substance for spinal cord cells is present in CM. However, the

304 neurite growth-promoting activity in CM was not specific for spinal cord cells. CM also poterttiated neurite growth from periphera13,5,6,10,16,'~z,25,zs and central neurons2,7,11, 25. Cell culture is a favorable system for the investigation of the effects of bic,logically active substances on cells. However, cultured cells are significantly affected by a variety of culture conditions such as culture medium, serum, substratum, etc. For this reason, the conditions under which studies of neurite growth are performed are crucially important. In this study, the effects of serum, tissue extracts, CM and culture substrata on neurite appearance from spinal cord explants were investigated. The results showed that neurite appearance was observed on polyornithine coating (PORN) even when any exogenous growth-stimulating factor was not present, but not on collagen coating. CM, serum, and tissue extract potentiated or induced neurite appearance, but their activities were dependent on the substrata. MATERIALS AND METHODS

Spinal cord cultures Spinal cords were dissected from 6- to 8-day-old chick embryos and cut transversely into small pieces in Eagle's minimum essential medium (MEM). Twenty to forty pieces were placed in a culture dish (35 ram, Falcon 3001) and preincubated with a small amount of MEM at 37 °C in a humidified atmosphere of 5 ~ CO2 and 95 ~ air for approximately 1 h to secure adhesion to the dish. Next, the medium was replaced by 2 ml of assay medium and the culture was maintained for 2-7 days in an incubator. Culture medium, tissue extracts, and conditioned medium The basic culture medium was Eagle's MEM (Nissui, Tokyo) (1.5 g/l NaHCO3). Fetal calf serum (FCS) was obtained from Flow Labs, McLean, VA. For preparation of tissue extracts, whole brain, pectoral muscle, heart, or liver of newborn chickens was homogenized with a glass homogenizer in 10 vols. (v/w) of 50 mM phosphate buffer, pH 7.4, containing 10 mM EDTA and 1 mM PMSF (phenylmethylsulfonyl fluoride, Sigma). The homogenates were centrifuged for 1 h at 10,000g. The super-

hates were used for the study. Protein levels in the extracts were determined by the method of Lowry et al. is with bovine serum albumin as the standard. Conditioned medium was obtained from cell cultures of the pectoral and thigh muscles of chick embryos. The muscles were dissected from 12- or 13day-old chick embryos and dissociated with 0.25 °/o trypsin in CaZ+-MgZ÷-free Hanks' balanced salt solution (BSS). About 3 × 107 cells were plated in a tissue culture flask (Falcon 3024, 75 cm 2) and cultured with 15 ml of MEM supplemented with 5°,,~ (v/v) FCS (5 ~ FCS-MEM). When the cells became confluent and myotubes were formed after 3 or 4 days of culture the medium was changed to fresh serum-free (SF) or serum-containing media for conditioning. CM was collected every 3-4 days up to 2 or 3 times from each culture. For SF-CM preparation, the cultures were washed with SF-MEM to remove serum before conditioning.

Dialysis and heat treatment SF-CM, FCS and tissue extract were dialyzed twice (1-day each) against 20 volumes of Earle's BSS. After dialysis, SF-CM was supplemented with amino acids and vitamins to reconstitute MEM. Some of the non-dialyzable fraction of SF-CM was concentrated 5 times with an Amicon ultrafiltration system (Model 52, PM 10 membrane). The dialyzable fraction of SF-CM was obtained by dialysis of MEM against SF-CM (10 ml of MEM per 100 ml of SF-CM for 2 days). The dialyzable fraction of FCS was obtained by dialysis of FCS against MEM (10 ml) of FCS per 100 ml of MEM for 2 days). FCS, tissue extract, and CM were heat-treated by incubation for 30 rain at 56 °C in a water bath. Before use, all assay media were sterilized by filtering through 0.22 # m Millipore filters. Collagen coating of dishes Collagen was extracted from rat tail tendons with 0.1 ~o acetic acid for 1 day 4 and non-dissolved material was removed by centrifugation for 30 min at 10,000 g. Collagen content was determined by the method of Lowry et al. 18 with type I collagen from calf skin as the standard. After preparation, the collagen solution was stored in a refrigerator at 4 °C and used within 1 month. Collagen solution concentrations were adjusted prior to coating (230 #g/ml in

305 most experiments) and 300/A was applied to the bottom of each culture dish. Collagen-coated dishes were dried in a dessicator under reduced pressure 9. Before use, the dishes were sterilized by ultraviolet irradiation.

PORN coating of dishes Coating with P O R N was performed according to the method of Varon et al. 2a. Dishes were incubated overnight with 1 ml poly-L-a-ornithine HBr (Sigma type I-C) (0.01-1.0 mg/ml in 0.15 M borate buffer, pH 8.4; in most experiments, 0.1 mg/ml). Next, dishes were washed 3 times with distilled water and dried in a dessicator.

Assay of neurite appearance from explants In explant culture, neurite can grow: (1) onto the non-cellular substratum; (2) upon the migrating non-neuronal cells; and (3) within the explant tissue. Only the first type of growth has been investigated in most experiments including the present one. This type of the growth has been called neurite growth or outgrowth "n the previous papers7,15,z0, z5 but will be described here as neur;te appearance in order to differentiate from type 2 and 3 grewth. For the assay of neurite appearance, the method of Obata and Tanaka 25 was used on a 2-day culture of cord explants. The number of single neurites or bundles of neurite was counted which extended over a distance of 300 # m from the edge of the explant. Each explant was graded on a scale of 0, 1 and 2 relative to the neurite number: 0, none (Figs. 1A and 5A); 1, 1-10 neurites; 2, over 10 (Figs. 1B and 5C, D). Usually the explants with scale 2 bore num6~ous (far over 10) neurites. In FCS-MEM many neurites were extended on the migrating non-neuronal cells (type 2 growth, Fig. 1A). Such migration in 2 d a y culture reached 100-250 # m but never exceeded 300 /~m. Therefore, such explant was graded 0. Mean score of neurite appearance was calculated on 30435 explants in 1-14 dishes. RESULTS

rites were observed on non-neuronal cells around the explants, but did not advance beyond the nonneuronal cells. Neurite appearance, however, was induced by addition of FCS or tissue extract to SFMEM or in CM on collagen coating (Fig. 1). The amount of collagen was crucial to neurite appearance. Fig. 2 shows the relationship between the amounts of coated collagen and the mean scores of neurite appearance in 1 0 ~ FCS-MEM. Neurite appearance was affected by amount of collagen ~Fig. 2). It was inhibited at higher collagen levels (over 706/tg protein/dish). At optimal collagen levels, the neurites were assumed to be tightly adhered to the substratum because they were curved and independently adhered to the collagen coating as in Fig. IB and C for CM. In the following experiments, 70/~g collagen was applied to each dish.

"

Collagen coated dishes Amount of collagen In SF-MEM and 2.5 ~ FCS-MEM (Fig. 1A) neu-

Fig. 1. Neurite appearance from spinal cord explants of 7day-old chick embryos cultured on collagen substratum for 2 days. A: 2.5~ FCS-MEM, score 0. B: 10~ FCS=CM, score 2. C: 2.5~ FCS-CM, score 2. The bar represents 300/~m for A and B, and 150/~m for C.

306

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Collagen (~,g/dish) Fig. 2. Effect on neurite appearance of collagen applied to the dishes as coating. Spinal cord explants from 6- to 8-day-old chick embryos were cultured for 2 days with 10 ~ FCS-MEM. Neurite appearance was calculated as the mean score 4S.E.M. for 42-149 explants.

Z

0

Serum

F C S i n d u c e d neurite a p p e a r a n c e f r o m spinal c o r d explants in a d o s e - d e p e n d e n t m a n n e r as shown in Fig. 3 ~open circles). The m a x i m u m effect was obt a i n e d with 10 ~ F C S . T h e action o f F C S was influenced b y slight changes in p H . N e u r i t e a p p e a r ance was greatest when 1 0 ~ F C S - M E M was buffered with 1.5 g/1 N a H C O 3 ( d a t a n o t shown). I n the following experiments, therefore, M E M was supplem e n t e d with 1.5 g/1 N a H C O 3 . T h e activity o f F C S was greatly r e d u c e d by dialysis (crosses) a n d was a l m o s t c o m p l e t e l y d e s t r o y e d b y h e a t t r e a t m e n t (56 °C, 30 min; Fig. 3, solid circles). T o determine the heat sensitivity o f the nondialyzable, high molecular weight ( H M W ) f i a c t i o n a n d the dialyzable, low m o l e c u l a r weight ( L M W )

I

2.5

I0

20

FCS (%) Fig. 3. Effects of FCS on neurite appearance from spinal cord explants cultured for 2 days. Concentrations of FCS in the culture medium or in the preincubation medium are on the abscissa. In the culture medium, FCS was native (©), dialyzed against Earle's BSS for 2 days (×), and heat treated at 56 °C for 30 rain (O). In another series of experiments collagencoated dishes were preincubated with 1 ml of MEM supplemented with FCS (concentrations on the abscissa) for 2 days and, after washing with MEM, the explants were cultured in SF-MEM (A) and 2.5 ~ FCS-MEM (~). Neurite appearance was calculated as the mean score 4- S.E.M. for 45-181 explants.

fraction o f F C S , the two fractions were separately h e a t - t r e a t e d (Table I). The H M W fraction was a b o u t h a l f as active as whole serum a n d its activity

TABLE I Relation between H M W and L M W fractions o f FCS

HMW-F, non-dialyzable fraction; LMW-F, dialyzable fraction; ~, v/v. Preparation

Mean score i S.E.M. per explant

No. explants tested

10~ 10~ 10~ 10~ 10~ 10~ 10H

0.81 0.43 0.02 0.11 0.86 0.18 0.87

105 262 108 120 205 118 200

FCS HMW-F of FCS Heated HMW-F of FCS LMW-F of FCS HMW-F of FCS 4- 10~ LMW-F of FCS Heated HMW-F of FCS q- 10% LMW-F of FCS HMW-F of FCS + 10~ heated LMW-FofFCS

5

± 0.06 ± 0.03 ± 0.01 ± 0.03 ± 0.04 ± 0.04 5_ 0.05

307 was lost following heat-treatment. The stimulating effect of the LMW fraction alone on neurite appearance was quite low, but was restored in the presence of the H M W fraction. The effectiveness of the LMW fraction-HMW fraction mixture decreased when the H M W fraction was heated, but was retained when the LMW fraction was heated. These results indicate that neurite appearance is affected by two fractions: a heat-labile H M W fraction and a heat-stable L M W fraction. Whether FCS exerts its effect by adsorption of active components to collagen or by free factors in the medium was investigated. Collagen-coated dishes were incubated with 1 ml of 2.5% to 20% FCS-MEM for 2 days and then washed with 2 ml of M E M 5 times before culture. Neurite appearance from spinal cord explants in SF-MEM in the pretreated dishes was observed (Fig. 3, solid triangles). For preincubation, the H M W fraction of FCS was similarly effective, but heat-treated FCS was not. The neurite appearance scores in SF-MEM were: 10% FCS, 0.57 (185 explants); 10% H M W fraction of FCS, 0.53 (163 explants); 10% heat-treated H M W fraction of FCS, 0.01 (126 explants). In FCStreated dishes, neurite appearance was greatly potentiated with 2.5 % FCS-MEM, (open triangles). These results suggest that some heat-labile, non-dialyzable component of FCS is adsorbed by collagen and induces neurite appearance. Tissue extract

Skeletal muscle extract alone had no effect on neurite appearance (not shown). In the presence of FCS (Fig. 4, open circles), however, the extract facilitated neurite appearance at an extremely low concentration (1 #g protein/ml). At higher concentrations, this effect decreased. The activity of the extract was heat-stable (crosses) and non-dialyzable (not shown). In contrast, the complementary factor in FCS was heat-labile as revealed by the ineffectiveness of a mixture of extract and heat-treated FCS (solid circles). Activity of extract was observed not only in skeletal muscle but also in heart, liver, and brain. No quantitative studies were performed on these and other tissues. Collagen-coated dishes were preincubated with skeletal muscle extract as in the case of FCS. Promotion of neurite appearance in pretreated dishes was

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0.1

0.3

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30

Muscle extract (Jug protein/rnl) Fig. 4. Effectsof skeletal muscle extract on neurite appearance from spinal cord explants cultured for 2 days. Concentrations of the extract for the culture or preincubation are on the abscissa. With non-preincubated collagen dishes the culture were performed in extract + 2.5 % FCS-MEM (©), extract + 10% heat-treated (56 °C, 30 rain) FCS-MEM (O), and heattreated (56 °C, 30 min) extract + 2.5 % FCS-MEM (×). With the collagen-coateddishes which were preincubated with 1 ml of MEM supplemented with skeletal muscle extract (concentrations on the abscissa) for 2 days and washed with MEM, the assay was done in 2.5 ~ FCS-MEM (zS). Neurite appearance was calculated as the mean score 4- S.E.M. for 30-80 explants.

observed with 2.5% FCS-MEM (open triangles), but not with SF-MEM. The active component in the extract appeared to be adsorbed by collagen and affect neurite appearance. At higher concentrations of preincubated extract, promotion of neurite appearance decreased. Conditioned medium

The CMs of heart and skeletal muscle of chick embryo were highly active in inducing neurite appearance from spinal cord explants as previously reported 25. The CMs of chick embryo brain cells and C6 cells, a rat glial cell line, were similarly active (unpublished observations). In the present investigation, the effects of skeletal muscle CM were analyzed. With CM, the onset of neurite appearance was more rapid than with FCS or a mixture of FCS and tissue extract. Some neurites extended as far as 300/~m from the explants within 12 h with CM. More than 1 day was required with other media to achieve similar lengths.

308 CM was prepared by culturing skeletal muscle cells in serum-free (SF-MEM) or serum-containing M E M (FCS-MEM). 2 . 5 - 1 0 ~ FCS-CM showed the high neurite appearance promoting activity (mean score, 1.46 for 2.5 ~ FCS-CM in Table II). SF-CM was also active, although it was less potent than FCS-CM. The activities of SF-CM and 2.5 ~o FCSCM were heat-stable. The higher score for FCS-CM was not simply due to the addition of FCS activity because: (1) 2 . 5 ~ heat-treated F C S - M E M was practically inactive; (2) the activity of 2 . 5 ~ FCSCM was not reduced by heat-treatment and 2.5 heat-treated FCS-CM was more active than SF-CM (Table II); (3) native FCS, H M W and heat-treated H M W fractions of FCS similarly potentiated the effect of SF-CM. The neurite appearance scores were: SF-CM, 0.30 (79 explants); SF-CM -t- 2.5 FCS, 0.97 (67 explants); SF-CM + 2 . 5 ~ H M W fraction of FCS, 0.97 (62 explants); SF-CM + 2.5 heat-treated H M W fraction of FCS, 0.92 (66 explants) for 1 day of culture. The activity of SF-CM was potentiated by addition of 2.5 ~ FCS to the levels attained with 2.5 ~ FCS-CM (Table II). These

results showed that a heat-resistant and non-dialyzable component(s) of FCS highly potentiated SFCM. Furthermore, it could be concluded that FCS interacted with CM to promote neurite appearance in spinal cord cultures rather than to induce more active CM by affecting muscle cell cultures. H M W and L M W fractions of SF-CM were obtained by dialysis (Table III). The H M W fraction retained only weak activity and the L M W fraction showed no activity. The activity of SF-CM was recovered almost completely by mixing 0.5 ml of 5-fold concentrated H M W fraction with 1.5 ml of the L M W fraction. This indicates that at least two components are required for neurite appearance in the presence of SF-CM as in the case of FCS. The effects of these two SF-CM fractions when combined with corresponding FCS fractions were investigated (Table III), The H M W fractions of SFCM was reactivated by the L M W fraction of 1 0 ~ FCS. Similarly, the activity of 10 ~ H M W fraction of FCS was potentiated by the L M W fraction of SFCM. When the H M W fraction of FCS was heattreated and then combined in a 10 ~ concentration

TABLE II Relation between C M and F C S

CM type; 2.5 % FCS: CM was prepared by culturing skeletal muscle cells with 2.5 % FCS-MEM; 2.5 % heated FCS: with 2.5 heat-treated FCS-MEM; serum free: with SF-MEM, %; v/v. C M type

2.5 % FCS 2.5 % FCS 2.5% Heated FCS Serum-free Serum-free

Treatment

Heat (56 °C, 30 min) ÷ 2.5 ~ FCS

Mean score i S.E.M. per explant

No. explants tested

1.46 -k 0.08 1.49 ± 0.08 1.12 4- 0.08 0.61 4- 0.03 1.42 4- 0.03

61 59 57 406 435

TABLE 1II Relation between H M W and L M W fractions o f S F - C M and F C S

HMW-F, non-dialyzable fraction; LMW-F, dialyzable fraction; %, v/v. l~reparation

Mean score 4- S.E.M. per explant

No. explants tested

SF-CM HMW-F of SF-CM LMW-F of SF-CM HMW-F of SF-CM ÷ HMW-F of SF-CM + LMW-F of SF-CM -LMW-F of SF-CM ÷

0.61 ! 0.03 0.11 -q- 0.02 0.00 4- 0.00 0.57 ± 0.07 0.69 4- 0.05 1.06 _k 0.05 0.00 ± 0.00

406 303 30 106 152 180 40

LMW-F of SF-CM 10700LMW-F of FCS 10~ HMW-F of FCS 10n heated HMW-F of FCS

309 TABLE IV Pretreatment o f collagen-coated dishes with S F - C M HMW-F, non-dialyzable fraction; LMW-F, dialyzable fraction. Incubated medium

Assay medium

Mean score ~: S.E.M. per explant

No. explants tested

SF-CM SF-CM H M W - F of SF-CM L M W - F of SF-CM

MEM 2.5 ~ FCS-MEM 2.5 ~ FCS-MEM 2.5 ~ FCS-MEM

0.03 0.73 0.69 0.09

291 304 81 97

with the L M W fraction of SF-CM, activity was not restored. Thus, both H M W and L M W fractions, regardless of their origin, were shown to be necessary to promote significant neurite appearance on collagen. Heat-treatment did not alter the effects of the L M W fractions of FCS and SF-CM, and H M W fraction of SF-CM, but destroyed that of the H M W fraction of FCS. Collagen-coated dishes were preincubated with SF-CM in the manner described previously for FCS and tissue extract. Promotion of neurite appearance in pretreated dishes was observed with 2.5 ~ FCSM E M , but not with SF-MEM (Table IV). Preincubation with the H M W fraction of SF-CM also promoted neurite appearance in 2.5 % FCS-MEM, but not with the L M W fraction of SF-CM. These results suggest that some H M W component of SF-CM exerts its effect on neurite appearance after adsorption to collagen as in the case of FCS and tissue extract, while the L M W fraction does not.

PORN-coated dishes Compared to collagen, P O R N appeared to be more favorable for neurite appearance. Adhesion of the explants to P O R N was much greater. Approximately 1 h was required for the explants to become firmly adhered to collagen. In contrast, only 5 min were needed for complete adhesion to P O R N in SFMEM. In SF-MEM on collagen, non-neuronal cells migrated out from the explants and neurites grew on only such cells. On P O R N , however, migration of non-neuronal cells was reduced markedly and neurite appearance was observed with a mean score of 0.37-0.64 (Fig. 5A and Table V). The numerous neurites were extended, reaching 2-3 m m in SF-MEM on P O R N after 5-7 days of culture (Fig. 5B).

-I- 0.01 q- 0.04 i 0.08 ± 0.03

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Fig. 5. Neurite appearance from spinal cord explants of chick embryos. The culture dishes were coated with PORN. A: explants were cultured for 2 days in SF-MEM (score 0). B." cultured for 5 days in SF-MEM. C: cultured for 2 days in SFCM (score 2). D" cultured for 2 days in 2.5~o FCS-MEM (score 2). The bar represents 150/~m for A, D and 300/*m for B, C. Very significant differences in the morphology of neurites on collagen and P O R N were observed. On collagen, neurites were straight and their shafts were smooth. These nettrites also tended to form bundles (Fig. 1C). On P O R N , neurites grew separately except in crowded areas. Along their shafts, each neu-

310 TABLE V Neurite appearance on P O R N

HMW-F, non-dialyzable fraction; LMW-F, dialyzable fraction; (1), (2), different lot of PORN was used. Preparation

Mean score ± S.E.M. per explant

No. explants tested

(1) MEM 2.5 ~ FCS-MEM 10~ FCS-MEM

0.64 ± 0.04 1.02 ± 0.04 1.25 i 0.04

273 246 277

(2) MEM SF-CM HMW-F of SF-CM LMW-F of SF-CM

0.37 1.51 0.50 1.24

145 235 229 113

± 0.04 ± 0.04 :k 0.04 zk 0.05

rite had many thick regions which appeared to be growth cones. Numerous lateral filodopia extended radially from these areas (Fig. 5D). Varying concentrations of P O R N solutions used for coating showed similar effects on neurite appearance at 0.03-1.0 mg/ml, but the effect with 0.01 mg/ml was greatly reduced. Different lots of P O R N also had somewhat different mean scores (Table V(1), (2)). Neurite appearance on P O R N was greatly facilitated by CM (Fig. 5C and Table V). In CM, neurites appeared much earlier and were more numerous. FCS also potentiated neurite appearance, but not to the same extent of CM. In FCS containing culture media, neurites tended to adhere to each other and formed networks on migrating non-neuronal cells around the explant. The number of neurites extending away from the explants decreased in older cultures. Skeletal muscle extract showed only weak effect on neurite appearance on PORN at concentrations of 0.2-125 #g protein/ml in the presence of serum. The effects of the H M W and LMW fractions of SF-CM on P O R N were quite different from those on collagen (Table V). The activity of the L M W fraction was almost equal to that of whole SF-CM. In contrast, the activity in the H M W fraction was quite low. DISCUSSION

In MEM with no supplement, neurite appearance from spinal cord explants was not observed on col-

lagen, but on PORN. CM and FCS highly potentiated neurite appearance. Two important aspects of neurite appearance from spinal cord explants were revealed: (1) spinal cord explants extended neurites spontaneously on PORN. This neurite appearance was slow in onset, but extensive; (2) a variety of substances such as culture substratum, CM, tissue extract, and FCS, affected neurite appearance. A number of previous reports have demonstrated t h a t C M 2,3,5-7,1°,11,16,22,25,28 and tissue extract 17,~0, ~1,24 facilitate neurite growth in culture. This activity was assumed to be exerted by a H M W substance(s) because it was retained after ultrafiltration or dialysis6,7,10A7, and was eluted in H M W fraction by gel filtration chromatographyU'2L In addition, serum has been assumed to have no significant effect on neurite growth. In the present study, it was found that not only H M W components but also a LMW component(s) found in CM and FCS significantly affected neurite appearance from spinal cord explants. The use of SF-CM for the experiments and SF-MEM as the control was instrumental in this determination. In a previous report zS, serum was conventionally included in the CM. Therefore, it is possible that the effects of serum were overlooked. Before presenting a discussion of medium effects, the role of the substratum should be considered. Neurite appeared from spinal cord explants in SFMEM on PORN, but not on collagen. Other remarkable differences between P O R N and collagen were that the adhesiveness of the explants to P O R N was much greater than to collagen and that migration of non-neuronal cells was much less prominent

311 on PORN. These factors will be related to difference in neurite appearance. Some components in HMW fractions of CM, FCS and tissue extract enhanced neurite appearance on collagen after deposition but not on PORN. It seems possible that this is due to change in adhesiveness of collagen. However, these depositable components were not identical. The FCS component was heat-labile and stimulated neurite appearance even with SF-MEM. On the other hand, components of SF-CM and extract were heatstable and potentiated neurite appearance with 2.5 ~ FCS-MEM, but not with SF-MEM. Higher levels of collagen and adsorbed extract were less favorable for neurite appearance (Figs. 2 and 4). The effect of PORN on neurite appearance, however, did not vary even when the ameunt used varied significantly. This suggests that the stimulating mechanism(s) of collagen and extract may be different from that of PORN. Less active migration of nonneuronal cells on PORN possibly facilitated neurite appearance but it seems unlikely that the neurite effect of FCS, CM and extract is indirectly exerted through depression of non-neuronal migration, because these substances did not affect the migration markedly (compare Fig. 1B with A and Fig. 5A to D). On collagen, when both the HMW and LMW fractions from either CM and FCS were included in the medium, neurite appearance was greater than when the fractions were used separately. Similar effects were observed with these fractions from FCS and CM. Corresponding fractions had equal effects, but did not share all the same components because their heat stability differed. The neurite appearance promoting activity of SF-CM was highly potentiated by heat-stable HMW component(s) in FCS. This suggests that the HMW fraction of FCS contains multiple components which stimulate neurite appearance on collagen because the collagen-adsorbable HMW component of FCS was heat-labile. On PORN, neurite appearance was potentiated most effectively by the LMW fraction of SF-CM. The LMW fraction of FCS also stimulated neurite appearance, but its activity was not as high as that of SF-CM. HMW fractions of SF-CM and FCS, and tissue extract had low neurite appearance scores. It was assumed that the HMW fraction of SF-CM contained only a collagen-adsorbable component;

however, the following findings did not support this assumption. In SF-CM pretreated collagen-coated dishes, potentiation of neurite appearance was observed with SF-CM or 2.5~o FCS-MEM, but not with LMW fraction of SF-CM. This suggests that neurite appearance on collagen with SF-CM requires the non-collagen adsorbable HMWcomponent. The results of this study further suggest that multiple components stimulate neurite appearance from spinal cord explants and that this activity is highly dependent on culture conditions. Under the most favorable condition (cultures on PORN), LMW fraction of SF-CM was sufficient to promote neurite appearance. The effects of HMW fractions were not revealed. Under moderately favorable condition (cultures on collagen), both LMW fractions and HMW fractions of SF-CM and FCS were necessary to promote neurite appearance. CMs of heart, skeletal muscle and glioma C6 cells have been shown to facilitate neurite growth from sympathetic, ciliary and dorsal root ganglion cells, retina and optic tectum~s. Neurite growth-promoting activity in CM was effective to a variety of neurons. However, it was not determined whether identical substances enhanced neurite growth from all neural tissues or whether CM contained multiple neurite growth-promoting factors. CM of skeletal muscle cells also has been reported to contain trace amounts of NGF 23. It is necessary to elucidate the active component(s) of CM under suitable bioassay conditions. ACKNOWLEDGEMENTS The authors wish to express their appreciation to Dr. T. Hayashi of Tokyo Medical and Dental University for his gift of collagen and to Yoshiko Aoki and Yukie Roppongl for their technical assistance. This work was supported by a Grant-in-Aid for the Encouragement of Young Scientists from the Japanese Ministry of Education, Science and Culture and Research Grants from the Naito Science Foundation (for 1980) and the Japan Medical Association.

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