Collagen and mucopolysaccharide biosynthesis in mass cultures and clones of chick corneal fibroblasts in vitro

DEVELOPMENTAL

BIOLOGY

21,

611-635 (1970)

Collagen and Mucopolysaccharide Biosynthesis Mass Cultures and Clones of Chick Cornea1 Fi bro blasts in Vitro’

in

GARY W. CONRAD' Department

of Biology,

Kline Biology Tower, Yale University, Connecticut 06520 Accepted

New Haven,

October 6, 1969

INTRODUCTION

'

Fibroblasts produce a number of well characterized extracellular products, including structural proteins and mucopolysaccharides. Different tissues in uiuo contain specific arrays of fibrous proteins (Seifter and Gallop, 1966) and mucopolysaccharides (Meyer et al., 1956) presumably because of epigenetic differences between their fibroblast populations. These tissue-specific differences have been difficult to demonstrate in fibroblasts in vitro for at least two reasons: (1) the morphological similarity of fibroblasts from diverse sources in embryos and adults and (2) the apparent tendency of other cell types to assume the fibroblast morphology under certain culture conditions. Because of the difficulty of obtaining and recognizing a pure fibroblast population, the degree of specialization of these cells both in uiuo and in vitro remains undetermined. For example, does each fibroblast of a given population make collagen as well as the polysaccharides characteristic of that population or do some fibroblasts make collagen while others make one or more of the polysaccharides? To investigate cell specialization in vitro one must have means of obtaining pure populations of cells and techniques for the precise identification of their differentiated end products. Isolation of pure populations of striated muscle (Hauschka and Konigsberg, 1966), ’ Supported by predoctoral fellowship (5-Fl-GM-32,877-02) from the United States Public Health Service and research grant GB-3265X of Dr. J. P. Trinkaus from the National Science Foundation. This work represents partial fulfillment of the requirements for the Ph.D. degree at Yale University and has appeared in abstract form [J. Cell Viol. 39, 28A (1968)]. * Present address: Department of Pediatrics, Wyler Children’s Hospital, University of Chicago, Chicago, Illinois 60637. 611

CONFXAD cartilage (Coon, 1966; Abbott and Holtzer, 1963), and pigmented epithelium (Cahn and Cahn, 1966) has allowed precise analysis of the biosynthetic capabilities of these cell types in vitro. The embryonic chick cornea contains fibroblasts which can be dissected free from the associated epithelium and mesothelium to give a fibroblast population with the degree of cell purity requisite for biochemical study. The tissue-specific extracellular products of the cornea include collagen, keratosulfate, and chondroitin 4-sulfate, and the accumulation of these products in the cornea during embryogenesis has been examined (Anseth, 1961; Coleman et al., 1965; Hay and Revel, 1969; Conrad, 1970). The stromal fibroblasts of the cornea have been implicated in the biosynthesis of these products (Coulombre, 1964; Coleman et al., 1965; Hay and Revel, 1969). Techniques of mass culture and cell cloning directly from the embryo (Konigsberg, 1963; Cahn et al., 1967) now make possible a direct in vitro examination of cell specialization in the elaboration of these compounds. Although Green and Hamerman (1964) analyzed collagen and hyaluronic acid synthesis in clones from an established aneuploid cell line, only five clones were examined and the cells were derived by outgrowth from a whole minced embryo rather than by dissection of a preexisting fibroblast population. Since no previous clonal study of freshly isolated fibroblasts had dealt with the biosynthesis of sulfated polysaccharides, it was desirable to perform analyses on a large number of clones from a tissue-specific fibroblast population such as that in the cornea. Other factors may modify what connective tissue cells synthesize in vitro and thereby complicate interpretation of clonal studies. For example, the precise role of the cornea1 epithelium (Teng, 1961; Wortman, 1961) and mesothelium (Jakus, 1956, Brim et al., 1966) in the biosynthesis of the extracellular matrix of the cornea remains unelucidated. Moreover, the possibility exists that in mass culture comrective tissue cells may synthesize different kinds of polysaccharides during different phases of their culture cycle (Nameroff and Holtzer, 1967). For these reasons, the in vitro analysis of fibroblast specialization presented in this paper was addressed to the following questions: (1) Which cell types of the cornea synthesize the specific structural proteins and polysaccharides found there? (2) At what stages of their culture cycle do these cornea1 cells engage in synthesis? (3) To what degree are clonal populations of cells specialized for the synthesis of certain end products of differentiation?

CORNEAL

FIBROBLASTS

MATERIALS

IN VITRO

613

AND METHODS

Tissue preparation and culture conditions. White Leghorn eggs (SPAFAS, Inc.; Norwich, Connecticut) were incubated for various periods of time in forced-draft incubators. All subsequent steps were carried out under sterile conditions. Corneas were excised from 14-day embryos, transferred to calcium-, magnesium-free saline G containing 10% (v/v) chick serum (CMF-saline), and trimmed free of all limbal tissue with iridectomy scissors. Trimmed corneas were digested with collagenase (CLS, 2.5 mg/ml in CMF-saline) for 10 minutes at room temperature and rinsed twice in CMF-saline. Sheets of loosened mesothelium and epithelium were gently scraped from the stromas. The stromas were then transferred to CMF-saline and rinsed twice; sheets of intact epithelium were treated similarly. Mesothelium was not studied. Scraped stromas were dispersed by incubation for 20 minutes in 8 ml of collagenase solution on a gyratory shaker (148 rpm) at 36.5OC. The liberated stromal fibroblasts and debris were centrifuged at 25’C for 12 minutes at 1700 g, resuspended in nutrient medium containing no embryo extract (NM w/o EE), and filtered through five layers of cheesecloth. Samples of the resulting cell suspension were removed to determine cell density and mean cell volume with a Coulter electronic cell counter; samples in a hemacytometer were photographed to record the percentage of clumped cells; other samples were diluted with NM w/o EE and used to inoculate culture dishes (Falcon plastic, 60 X 15 mm, No. 3002) containing 3 ml of complete nutrient medium (NM). Dishes were incubated at 36.5’C in an atmosphere of 5% COz-balante air. Medium was changed three times per week by removing 1 ml and replacing it with 1 ml of fresh NM. After the cells were suspended in a solution of trypsin and EDTA (Cahn et al., 1967), the number of cells per dish and their mean cell volume was determined. The plating efficiency of freshly isolated fibroblasts was determined by plating the cells at low densities (20-200 cells/dish), incubating the dishes for lo-21 days with regular changes of medium, and counting the resulting colonies after staining. The culture medium used was F-10 (Ham, 1963) containing amino acids and pyruvic acid at twice the final published concentrations (Coon, 1966; Cahn et al., 1967), 5 or 10% (v/v) fetal calf serum, 0.5% bovine serum albumin, 50 units/ml sodium penicillin G, and embryo

614

CONRAD

extract. The lots of fetal calf serum employed were those that produced the highest plating efficiencies of freshly isolated cornea1 fihroblasts in NM w/o EE containing 15% fetal calf serum. Embryo extract was made according to Coon (1966) except that g-day embryos were used and the hyaluronidase digestion was performed for 1 hour. This EEso was used directly as “whole EE” or fractionated at 4OC on Sephadex G-25 into “light” and “heavy” fractions. After correcting for dilution, each of these EE preparations was mixed in varying concentrations with NM w/o EE to yield complete NM. Complete NM was stored at 4°C for no more than 2 weeks, and embryo extract preparations were kept at - 87°C for shorter periods. Media will be designated as follows (Coon, 1966): NM/H-2 represents NM containing the same concentration of “heavy” molecular weight material as is present in 2% whole EEXJ (v/v). Conditioned medium (CM) was made from NM/H-2 according to Rubin (1966), filtered through Millipore filters (0.45 P), and mixed in varying proportions (v/v) with unconditioned NM/H-2. Except where noted, all cloning was done in 60% CM, where the concentrated CM was collected from saturation phase cultures. Solutions were sterilized by pressure filtration through washed Millipore filters (Cahn, 1967). Incorporation of mdioisotopes by cells in vitro. Samples consisting of four untorn sheets of comeal epithelium were transferred to 1.0 ml of NM/H-2 containing 2 &X/ml of L-proline-14C (uniformly labeled) trapped beneath a glass coverslip the four comers of which had been heated and bent down 2 mm to form a chamber in the bottom of a petri dish. After varying periods of time, incubation mixtures consisting of labeling-medium plus tissue were analyzed for collagen synthesis as described below. Mass cultures of stromal fibroblasts were incubated for varying periods of time in NM/H-2 containing one of the following radioactive precursors (2 &i/ml): Naz35S04, n-glucosamine-6-3H, n-glucosaminel-‘“C, or r,-proline-14C (uniformly labeled). Unless otherwise stated, samples consisted of the labeling medium together with the cells scraped from the dish with a rubber policeman. All samples were stored at -2OOC until analyzed. Controls consisted of labeling medium and cells frozen at zero time. Macromolecular biosynthesis in single colonies of stromal fibroblasts was detected as follows. Dishes containing 60% CM were each inoculated with 35-40 cells and incubated for 24 hours. The positions of individual attached cells and cell clusters were marked on the dish bottom, and the dishes were reincubated for 2 or 3 weeks with regular

CORNEAL

FIBROBLASTS

IN VITRO

615

changes of medium. The fate of each identified cell or cell cluster was then recorded, as were any colonies arising from previously undetected cells. Resultant colonies, which were of varying size, cell density, and morphology, were photographed and incubated with radioistotopes in the following manner. A glass ring (11 mm i.d. X 16 mm o.d. x 9 mm) was placed around each colony and secured to the dish surface with silicon grease. Each ring was then filled with 60% CM (0.9 ml) containing either L-proline-14C (uniformly labeled) + D-glucosamine-6-3H or L-proline-3,4-“H + Na235S04, with each precursor present at a concentration of 5 &i/ml. Dishes containing the ringed colonies were reincubated, with no medium changes, for 1, 2, or 3 weeks. Controls consisted of labeling medium incubated for the same length of time in the absence of cells. After suspension with trypsin-EDTA solution, the cells from individual colonies were combined with the labeling medium from their respective rings to constitute individual samples. After two freeze-thaw cycles, each sample was divided into two equal portions, one for collagen analysis and the other for polysaccharide analysis by Procedure B (see below). Analysis of labeled collagen and polysaccharide. Collagen was extracted according to Lukens (1965)) except that after ether extraction the hot trichloroacetic acid supernatant fluids were dialyzed for 4-7 days against distilled water or 0.05 A4 Na2S04 in the cold and then lyophilized. Residues were either hydrolyzed or divided into two equal portions, one for digestion with collagenase (CLSP-A, 250 rg/ ml) according to Lukens (1966) and the other for control incubation with buffer. After incubation for 7 hours, samples were dialyzed for 4 days against distilled water, concentrated, and hydrolyzed. Proline, hydroxyproline, and other labeled compounds were resolved by elution from columns of Dowex 50 resin (Conrad, 1970): sulfate emerged in fractions 3 and 4, n-glucosamine and n-galactosamine in 6-9, Lglutamic acid in 10 and 11, hydroxy-L-proline in 12-20, and r,-proline in 25-38. No radioactivity was detected in fractions 21-23. The labeled polysaccharides were purified by Procedure A or B of Conrad (1970), except that selected ether-extracted residues were divided into two equal portions, one for hyaluronidase digestion (293 NF units/ml) according to Mathews and Inouye (1961) and the other for control incubation with buffer. After incubation for 24 hours (in the presence of toluene), samples were dialyzed for 5 days against distilled water or 0.05 M NaS04, concentrated, and chromatographed by Procedure B. Chemical assays. Amino acids in column eluates were detected with

616

CONRAD

1% ninhydrin in glacial acetic acid. Amino sugar was assayed according to Svennerholm (1956). Inorganic sulfate was determined according to Lloyd (1959). Radioactivity was measured in a Packard liquid scintillation counter for 50 or 100 minutes, using Bray’s solution (Bray, 1960). Corrections were made for the decay of 35S. Radioactivity in control samples was subtracted as background. Muter&. D-Clucosamine-l-‘4C (8.9-13.5 mCi/mmole), n-glucosamine-6-3H (238 mCi/mmole), L-proline-‘4C (uniformly labeled, 204219 mCi/mmole), L-proline8,4-3H (4.86 Ci/mmole), and Na235S04 (260-633 mCi/mmole) were obtained from New England Nuclear Corporation. Collagenase (CLSP-A and CLS, each at 150 units/mg) was purchased from Worthington. Ovine testicular hyaluronidase (Type II, 440 NF units/mg) was purchased from Sigma. RESULTS

Growth Characteristics

of Corned Fibroblasts

in Vitro

The fibroblasta of the chick cornea had not been studied in isolation free from other cell types. Thus it was important to examine their growth characteristics in some detail. To verify that cultures consisted only of cornea1 fibroblasts, scraped stromas were fixed, embedded, sectioned, and stained. No adhering mesothelial, epithelial, or limbal cells were seen (Fig. 1). Using such cultures of cornea1 fibroblasts, control experiments indicated that (1) rate of growth, increase in mean cell volume, and fmal saturation density of mass cultures as well as the plating efficiency of dilute suspensions were identical for populations grown on either Pyrex glass or Falcon tissue culture plastic dishes. (2) Dishes of either type containing NM/H-2 could be preincubated for as long as 24 hours prior to inoculation with dilute cell suspensions with no effect on the plating efficiency. Cell growth on collagen gels was therefore not examined. (3) Cells in confluent mass cultures or in clones could be surrounded by a glass cloning ring filled with 60% CM and incubated without medium changes for as long as 3 weeks without deleterious effects on the cells. Small clones rarely grew to dense populations inside the rings, but large clones often attained confluency within such areas. Clones were grown in media containing various preparations of embryo extract. Low concentrations of the H-fraction from embryo extract had little effect on clonal growth, but high concentrations of H-fraction and all concentrations of whole and light fractions tested caused a decrease in plating efficiency (Table 1). Because NM/H-2

FIG. 1. Scraping control. Sections were stained with Weigert’s acid iron chloride hematoxylin. (la) Cross section of 14-day cornea after dissection and trimming, but before scraping. Note intact epithelium and mesothelium and absence of any limbal tissue. (lb) Cross section of 14&y cornea after dissection, trimming, and scraping. Note absence of epithelium, mesothelium, and limbal tissue. Only cornea1 stroma fibroblasts remain. Bright-field. x 50.

was already being used successfully for mass cultures, it was used as the base medium for cloning as well. The effect of conditioned medium (CM) on the growth of clones was also examined. Concentrated CM was collected from mass cultures of cornea1 fibroblasts grown in NM/H-2 during both logarithmic and saturation phases of growth and diluted to varying proportions with unconditioned NM/H-2. Cells suspended in NM w/o EE were inoculated at 200 cells per plate and allowed to grow for lo-12 days. Clones were fixed, stained, and classified arbitrarily as either “small” clones (50 cells to clones 2 mm in diameter) or “large” clones (those larger than 2 mm in diameter). As shown in Fig. 2 the presence of CM derived from either logarithmic or saturation phase cultures increased the plating efficiency of the cornea1 fibroblasts. At concentrations of 60-SO%, CM markedly increased the percentage of clones which were “large.” Therefore 60% CM from saturation phase cultures was selected for cloning.

618

CONRAD TABLE 1 EFFECF OF DIFFERENT CONCENTRATIONS AND KINDS OF EMBRYO EXTRACT ON PLATING EFFICIENCY OF CLONES Media” Experiment w;zE

H-O.5

H-l

H-2

H-3

H-4 W-2

L-l

L-2

I Average number clones/ dish Plating efficiency (%)

20b

13

19

8

11

7

5

7

4

10

7

10

4

6

4

3

4

2

Average number clones/ dish Plating efficiency (%)

11’

13

10

7

1

0

2

2

0

6

7

5

4

0.5

0

1

1

0

II

’ H = heavy EE fractions, L = light EE fractions, W = whole EEso. ’ Each number represents an average of 5 dishes. Inoculum = 190 cells per dish for each experiment. Dishes in experiment I were incubated for 12 days; those in experiment II for 21 days.

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10

20

30

40

50

60

70

60

90

100

8

% OF CONDITIONED

MEDIUM

ap

IN NM/H-P

FIG. 2. Effect of log phase and saturation phase conditioned medium on plating e5ciency and clone size. Solid lines represent log phase; dotted lines, saturation phase. Open circles represent plating efficiency; crosses, percentage of counted clones which were “large.” Each point is an average of four dishes.

CORNEAL

FIBROBLASTS

IN

619

VITRO

Colonies were derived by plating freshly isolated cornea1 fibroblasts at low cell density. To demonstrate that these colonies were in fact clones it was necessary to establish not only that the inoculum population consisted primarily of single cells, but also that such single cells would grow into clones. In four different experiments samples of the original, undiluted cell suspensions were examined in the hemacytometer, photographs made of a total of 40 different fields, and tracings made of the enlarged images of each negative. A total of 5034 colony-forming units (a single cell or any aggregate of two or more cells) was examined. Of these 4762 (94.6%) were unequivocally single cells, 253 (5.02%) were aggregates of two cells (doubles), and 19 (0.38%) were triples. Only one aggregate of more than three cells was seen (four cells). The percentages of doubles in these four experiments were 5.5, 5.3, 4.8, and 4.1% while the percentages of triples were 0.98, 0.46, 0.24, and 0%. Therefore the percentage of cell clusters ranged from 4.1 to 6.5% of the undiluted inocuhrm population. In all cloning experiments the locations of attached single cells were marked 12-24 hours after their inoculation in uitro, and their growth was recorded after varying lengths of time in culture. The single cells gave rise to clones containing varying numbers of cells (Fig. 3). Of the 468 single cells located, 57 (12%) grew into clones of more than 50 cells and 28 (49%) of these were “large” clones (Table 2). In addition to the single cells, any cell clusters found after inoculation were also marked. In these five experiments, a total of 11 such cell clusters were found and 8 of these subsequently gave rise to colonies (Table 2). The plating efficiency of stromal fibroblasts in 60% CM was approximately 13% (Fig. 2), a value close to the percentage of identified single cells giving rise to clones in the five experiments described (12%). Since the percentage of cell clusters in the initial inoculum averaged only 6%, at least half and probably more of the colonies arising from cells plated at low density grew from single cells and were therefore true clones. Collagen and Mucopolysuccharide blasts in Vitro

Biosynthesis

by Coneal

Fibro-

Experiments were conducted to establish what percentage of polysaccharide synthesized was associated with cell layers compared to the amount released to the medium. Mass cultures which had just reached confluency (7 days in culture, 1.8 X lo6 cells/dish) Mass cultures.

FIG. 3. Growth of two clones. Left, clone 99-11/A. Right, clone 99-13/A. (3a and 3d) After 1 day of culture. (3b and 3e) After 4 days of culture. (3c and 3f) After 13 days of culture. Phase contrast. X 155.

620

CORNEAL

FIBROBLASTS

621

IN VITRO

TABLE 2 PLATING

EFFICIENCY

AND CLONE

CELLS

SIZE FROM IDENTIFIED

AND CELL

SINGLE

CLUSTERS

Experiment number 1

2

3

4

5

Total

101” 12

101 13

59 5

99 11

108 16

468 57

6

11

3

7

1

28

(1,

(:I

Single cells

Number identified Number which grew into clones Number which grew into “large” clones Cell clusters Number identified (numbers of cells/cluster) Number which grew into colonies of more than 50 cells Number which grew into “large” colonies

c&3”,

3 (2, 4, 6)

11 c&2,“,,

4)

1

3

0

1

3

8

0

2

0

1

3

6

’ Totals from twenty dishes in each experiment.

POLYSACCHARIDES

TABLE 3 CELL LAYERS

IN RNSED

Precursor Glucosamine-6-‘H %O,‘~ 35so,2-

Chondroitin sulfateb Keratosulfate’ (fraction 1) Chondroitin sulfate’ (fraction 5)

AND MEDIUM

Cells + medium”

Cells alone

Medium” alone

12,228 358 119

5368 81 39

6065 173 91

’ Medium = labeling-medium + three cell layer rinses with isotonic saline. bAverage counta per minute above background from six samples representing only polysaccharide precipitated with cetylpyridinium chloride by Procedure A. ’ Average counts per minute above background from three samples fractionated by Procedure B.

were incubated with 35S042- or glucosamine-6-3H (2 &i/ml) in NM/H-Z for 8 hours. In one-half of the samples cells, rinses, and labeling-medium were combined in single fractions while in the remaining samples labeling-media and cell layer rinses were pooled and analyzed separately from cells alone. Polysaccharides were isolated

622

CONRAD

by Procedures A and B. The results suggested that labeled polysaccharide was present in the medium as well as in rinsed cell layers (Table 3). In subsequent experiments, cells and labeling-media were pooled. The incorporation of glucosamine&“H and 35S042- into polysaccharide was studied by incubating these precursors with mass cultures in mid-log phase for varying lengths of time. Labeled polysaccharide was precipitated and measured by Procedure A. Sulfate incorporation per lo6 cells was linear for at least 16 hours while the incorporation of radioactivity from glucosamine accelerated with time. Similar patterns of incorporation of these labeled precursors were observed during the incubation of whole corneas in shaker flasks (Conrad, 1970). To determine whether macromolecular biosynthesis could occur in both dividing and nondividing cell populations, mass cultures were inoculated at lo5 cells/dish and allowed to progress through a culture cycle. Throughout all phases of the cycle only fibroblastic cells were seen (Fig. 4). No epithelium-like cell arrangements were ever observed. During six &hour intervals of the culture cycle selected dishes of cells were incubated with glucosamine-1-“C, 35S042-, or proline-14C (Fig. 5), and radioactivity incorporated into collagen and polysaccharide was measured. Data in Table 4 show that labeled, nondialyzable hydroxyproline was formed during log and saturation phases of growth. When labeled material from log phase cultures was treated with collagenase (CLSP-A), all the detectable radioactivity in hydroxyproline and 2550% of that in proline became dialyzable. These data demonstrate that both collagen and noncollagenous protein are synthesized throughout the culture cycle. Labeled polysaccharides were detected in the mass cultures during each labeling period of the culture cycle. Representative patterns of sulfate and glucosamine labeling during the sixth period are shown in Fig. 6. Radioactivity in fractions eluted from cellulose columns by Procedure B indicated the presence of sulfated polysaccharides in three areas: fractions 1, 3, and 5-7. Radioactivity from glucosamine1-14Cappeared in the same fractions and also in fraction 2. Similar patterns were seen during the five preceding labeling periods. The glucosamine pattern matches that seen in uiuo and in whole corneas in shaker flasks (Conrad, 1970). These experiments suggest that both dividing and nondividing cell populations synthesize keratosulfate (fraction l), low-sulfated chondroitin sulfate (fractions 2 and 3), and highly sulfated chondroitin sulfate (fractions 5-7).

FIG. 4. Cells seen during the culture cycle described in Fig. 5. (4a and 4b) Log phase, 66 and 77 hours of culture, respectively. (4c and 4d) Saturation phase, 151 hours of culture. Phase contrast. x 195. 623

624

CONRAD

J I

-=CELL -

NUMBER =LAEELLING PERIOD

-

=RELATIVE MEAN CELLVOLUME

0 A .

I

I

I

50

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OF

periods, cell number,

OF L-PROLINE-"C

Labeling

Period

,

150

,

and relative

BY MASS

mean cell volume during a cul-

4 CULTURES

DURING A CULTURE

1 Cagphase) 5 6

(Saturation (Saturation

phase) phase)

CYCLE

Total M-minute counts above background per 1 X 10” cells Proline

2 (Log phase)

,

200

INCUBATION

TABLE INCORPORATION

1

100

HOURS

FIG. 5. Labeling ture cycle.

20 IO

39,945” 31,334 5,871 14,071

Hydroxyproline 446 309 170 323

‘Each number represents an average of two samples. No radioactivity was detected in fractions l-11, indicating insignificant conversion of proline to glutamic acid and glutamine. Uniformly labeled Lproline-“C was used.

CORNEAL

93 16,000 2 8 0

FIBROBLASTS

IN

625

VITRO

MOO -=

14,000

S=O:

INCORPORA

7000

D

6,000

5 & ::

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8.000

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1.000

8 9 s r

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5 8 ?I w

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2 3 FRACTION

4 5 NUMBER

6

F

7

into polysaccharides durFIG. 6. Incorporation of ?‘5Oa’- and n-glucosamine-l-‘4C ing labeling period 6 of a culture cycle. Cultures were incubated with labeled precursors for 8 hours. Polysaccharides were fractionated by Procedure B.

The amount of radioactivity incorporated into the three classes of polysaccharides during the six labeling periods of the culture cycle was normalized with respect to cell number. The data suggested that during the first five periods the rates of glucosamine incorporation fell regularly to approximately 25% of the initial value, then increased sharply during the sixth period (Fig. 7), as illustrated for highly sulfated chondroitin sulfate. The changes in the rate of sulfate incorporation during the culture cycle differed from those of glucosamine, except with respect to the sharp increase in incorporation during the sixth period. To compare the rate of sulfation with that of the synthesis of the amino sugar-containing polymer, the “5S042- incorporation per lo6 cells was divided by the corresponding normalized value for glucosamine-14C incorporation during each particular labeling period for each class of polysaccharide. The “5S/‘4C incorporation ratios plotted in Fig. 8 for keratosulfate (fraction l), highly sulfated chondroitin sulfate (5-7), and all chondroitin sulfates (2-7) followed from this comparison. They suggest that the degree of sulfation of chondroitin sulfate rises as the cornea1 fibroblasts change from a dividing to a nondividing population. The degree of sulfation of keratosulfate may rise, although this is still under investigation. These

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FIG. 7. Incorporation of ?30,*- and n-glucosamine-1-“C into polysaccharide of group (#5 + 6 + 7) during a culture cycle. After the title, read the same legend as Fig. 6. Only incorporation into highly sulfated chondroitin sulfate (fractions 5-7) is shown.

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= #

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1

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FIG. 8. Changes in the ““S/“C ratio in polysaccharide fractions during a culture cycle. After the title, read the same legend as Fig. 6. Numbers in parentheses refer to fractions from F’rocedure B. 626

CORNEAL

FIBROBLASTS

IN VITRO

627

data corroborate the previous correlation between cessation of fibroblast division and increased sulfation of the chondroitin sulfate synthesized by corneas in uioo (Conrad, 1970). In both systems the hypothetical changes in the degrees of sulfation must remain open until sufficient amounts of polysaccharides are isolated to allow chemical analysis. Clones. Portions of confluent mass cultures of stromal fibroblasts were surrounded by a glass cloning ring filled with one of the doublelabeled 60% CM media previously described. Radioactive hydroxyproline was detected in the collagen fractions, and the types of radioactive polysaccharides recovered were identical to those isolated from mass cultures of cornea1 fibroblasts. The levels of radioactivity in collagen and polysaccharide were comparable to those subsequently seen in individual large clones. A total of 68 clones derived from plating stromal fibroblasts at low cell density were examined to determine whether each clone could make both collagen and all of the cornea1 polysaccharides. Clones were individually incubated with double-labeled 605 CM inside glass cloning rings. The radioactivity subsequently recovered in collagen and polysaccharide was directly proportional to the size of the clone. When sufficient labeling occurred (67 out of 68 clones), the pattern of labeled polysaccharide produced by each clone was comparable to that observed in mass cultures. The labeling of the polysaccharides by sulfate and glucosamine in two representative clones is shown in Fig. 9. Digestion of such preparations with testicular hyaluronidase indicated that all the radioactivity in fractions 2-7, but none of the radioactivity in fraction 1, became dialyxable. Comparison of these data with the elution profiles of polysaccharide standards suggest that each clone can make low- and highly sulfated chondroitin sulfate and keratosulfate. In 49 of 66 clones sufficient radioactivity was recovered in the collagen fraction to allow detection of radioactive hydroxyproline. Additional evidence that the radioactivity in the hydroxyproline fractions represented only hydroxylated proline, rather than another amino acid, followed from a plot of radioactivity in hydroxyproline against that in proline for clones labeled either with prohne-“C (uniformly labeled) or proline-3,4-“H (Fig. 10). One line describes data from one “C experiment; the other, data from two ‘H experiments, where the slope of each line represents the ratio hydroxyproline/ proline. Hydroxylation of proline-‘4C results in no change in specific activity. On the other hand, approximately one-half of the tritium

628

CONRAD

S%~INCORPORATION CLONE 103-6/l - =GLUCOSAMINE-6-H INCORPORATION. CLONE 105-6/1-Z

I I

3

2

IW

FIG. 9. Incorporation of %01’thesized by two clones.

4

6

5

7

FRACTION

NUMBER

and n-glucosamine-6-3H into polysaccharides syn-

Ii3 y=O.O269

I 2 TOTAL

4 50 MIN

X

I

6 COUNTS

8

ABOVE

10 BACKGROUND

I

12

I I _1 PRO LI NE

x 1O-3

FIG. 10. Hydroxypmline/proline incorporation ratios in collagen fractions from clones. Open circles represent samples labeled with pmlme-“C (uniformly labeled). Crosses represent samples labeled with proline-3,4-“H. Equation of each regression line was calculated by least-squares analysis.

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atoms in the 3,4 positions of the tritiated proline are lost during hydroxylation (Stone and Meister, 1962; Goldberg and Green, 1967) and the specific activity of the tritiated hydroxyproline becomes one-half that of the proline-3,4-3H. If in the present work, the radioactivity found in the hydroxyproline fraction was due principally to hydroxyproline derived by the hydroxylation of the administered radioactive proline, then the slopes of the two constrained regression lines (yintercepts = 0) obtained by least squares analysis should differ (Fig. 10). In contrast, if such radioactivity represented another ammo acid, the lines should be indistinguishable, for no loss of tritium would have occurred. Indeed, under the null hypothesis of no difference between the slopes of the regression lines, Pr { 1 &I ) >_ 2.74) = 0.01, where the observed t = 3.10, thereby showing the difference to be highly significant. Figure 10 presents data for 35 clones from three separate experiments where the treatment of collagen samples was identical. Dissimilar extraction procedures in separate experiments could alter the hydroxyproline/proline ratios. Nevertheless, the theoretical hydroxyproline/proline ratio of approximately 2 : 1 between ‘*C- and “H-labeled collagen, respectively, should be independent of labeling time, absolute isotope concentrations in the medium, clone size, counting efficiencies of “H and ‘*C, and presence of even large amounts of proline-labeled noncollagenous protein. In summary the data indicate that each clone hydroxylated some of the administered radioactive proline, suggesting that collagen synthesis occurred. Comeal epithelium. The possible synthesis of collagen by cornea1 epithelium from 1Cday corneas was studied by incubation with proline-14C. Table 5 shows the incorporation of radioactivity from proline into hydroxyproline, evidence which suggests that cornea1 epithelium at this age can synthesize collagen. No radioactivity was found in Dowex fractions l-11, indicating that the conversion of proline to glutamic acid and glutamine was negligible. TABLE INCORPORATION

Incubation

OF L-PROLINE-‘%

time (hours):

Hydroxyproline” Proline”

5 BY CORNEAL. EPITHELIUM

20

40

64

224 6665

299 9595

393 14,683

’ Each number represents an average of the total 50-minute samples. Otherwise, read the same legend as Table 4.

counts in each of two

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CONRAD DISCUSSION

The stromal fibroblasts may be the principal biosynthetic cell type of the cornea in Go, for when they are grown in vitro they appear to make collagen, keratosulfate, and low- and highly sulfated chondroitin sulfate, the end products of differentiation in the cornea. The comeal epithelium also synthesized collagen, a fact in agreement with the morphological evidence of Hay and Revel (1969) and studies of established epithelial cell lines (Goldberg and Green, 1968). The procedures used here to identify such extracellular products are based on certain assumptions. First, in the absence of elastin it was assumed that hydroxylation of peptide-bound proline and susceptibility of such material to collagenase degradation served to demonstrate collagen synthesis. Second, the elution of polysaccharides from calibrated columns of cellulose by Procedure B separated keratosulfate (fraction 1) from chondroitin sulfate (fractions 2-7) and distributed the latter according to its degree of sulfation. Resistance of material in fraction 1 to degradation by testicular hyaluronidase together with the susceptibility of material in all later fractions supported these identifications. This method, however, assumes negligible synthesis of sulfated glycoproteins, which might not be distinguished from keratosulfate. A study of macromolecular biosynthesis by stromal fibroblasts during the culture cycle in vitro was a necessary prerequisite to a clonal analysis. If logarithmic and saturation phase mass populations made different kinds of polysaccharides, then clones containing different proportions of dividing and nondividing cells might be erroneously described as different clonal types. Indeed, the data presented here suggest that the synthesis of collagen and all three cornea1 polysaccharides can occur in both dividing and nondividing cornea1 fibroblast populations in vitro. The observations of collagen synthesis are in agreement with previous studies of Davies and Priest (1967). Moreover, Pawelek (1969) observed synthesis of sulfated polysaccharide by chondrocytes in vitro during all phases of the culture cycle. Nameroff and Holtzer (1967), on the other hand, detected synthesis of sulfated polysaccharides by chondrocytes in vitro only during saturation phase. Pawelek suggested that the discrepancy in their results might be due to the effect of different culture conditions on biosynthesis. In the present study, the lack of agreement may be due to a difference in cell type as well as a difference in culture conditions. Polysaccharide appeared to be synthesized at different rates dur-

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ing different phases of the culture cycle, showing a regular decline throughout log phase followed by a sharp increase after confluency was attained. Such changes in the rate of synthesis have also been noted in hyaluronic acid production by fibroblasts in vitro (Davidson, 1964; Hamerman et al., 1965) and in chondroitin sulfate synthesis by chondrocytes (Nameroff and Holtzer, 1967). These changes may be related to the state of differentiation of the cells examined. In the present study for example, cornea1 fibroblasts were removed for culturing on day 14 of embryogenesis when, according to Hay and Revel (1969), they were not completely differentiated morphologically. Their continued differentiation in vitro might be responsible for changes in the rate of biosynthesis. In addition, the transient reduction in the rate of synthesis of collagen and polysaccharidea during labeling period 5 may be a manifestation of contact inhibition of macromolecular synthesis (Levine et al., 1965). This culture cycle event is paralleled by an in uiuo diminution of synthetic rates prior to day 14 of embryogenesis (Conrad, 1970). On day 14 of cornea1 development in uiuo, division and immigration of stromal fibroblasts stop and, although the cells appear to be well separated from one another, Hay and Revel (1969) have demonstrated intimate contacts between them through long filopodia. Consequently, both in uiuo and in vitro contact inhibition may affect macromolecular biosynthesis. The fibroblast population of the chick cornea1 stroma appears to be homogeneous, for each clone grown from it can make collagen, keratosulfate, and chondroitin sulfate. This conclusion is subject to a number of qualifications which apply as much to earlier studies of clones as to the present one. (1) Comeal fibroblasts were cloned only from 14-day embryos. It is not known whether cells from the same population at different embryonic ages would exhibit different growth requirements and patterns of biosynthesis. The only data concerning the differentiation of this particular fibroblast population come from the elegant morphological study of Hay and Revel (1969). (2) Only about 13% of the single cells plated grew into clones large enough to analyze, leaving 87% of the in uiuo fibroblast population unexamined. Similar cloning efficiencies have been observed in previous studies (Green and Hamerman, 1964; Hauschka and Konigsberg, 1966). The unexamined portion of the population could represent damaged cells or those whose stage of differentiation precluded their division in vitro, similar to the phenomenon seen in myogenesis (Okasaki and Holtzer, 1965). However, the fact that wounding the adult cornea in uiuo results in reactivation of mitosis in almost all adjacent

632

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stromal fibroblasts (Weimar, 1960) argues against such an irreversible (3) In no case presented here is the biosynthetic history of a particular fibroblast known. It is not known whether the progenitor cell of any analyzed clone ever synthesized collagen or polysaccharides before its progeny began these processes in uitro. Jn contrast, the cell giving rise to a clone of pigmented retinal epithelium contains pigment granules, the expected end product of differentiation (Calm and Calm, 1966). (4) The degree of differentiation arising within the clones is not known. The clonal data are a valid assay of homogeneity in a population only to the degree that the clonal progeny of a single cell reflect the pathway of differentiation of that cell. Variation of phenotype within clones has been reported by Franks and Dawson (1966) and variations in clone size from supposedly homogeneous populations have been noted previously (Nias and Lajtha, 1965). Despite these qualifications, the analysis clearly suggests that individual clones of cornea1 stroma fibroblasts in vitro, and therefore perhaps single cells, are capable of synthesizing more than one end product of differentiation. The possibility that one of these products was not synthesized because of variations in the proportion of dividing or nondividing cells in different clones was eliminated by the mass culture data. If a clone was of sufficient size, the whole spectrum of polymers appeared. Although there may be variation in the proportions of the compounds made by each clone, the present data do not permit such a quantitation.

loss of proliferative capacity during differentiation.

SUMMARY

Collagen and mucopolysaccharide biosynthesis by cell populations of the chick cornea have been studied in vitro. Stromal fibroblasts from lkday embryos were grown as mass cultures and clones; cornea1 epithelium was isolated as intact sheets of cells from embryos of the same age, Cultures were incubated with radioactive precursors of collagen or mucopolysaccharides. Cornea1 epithelium hydroxylated a portion of the administered proline and therefore appeared to synthesize collagen. Mass cultures of stromal fibroblasts were incubated with the labeled precursors at various times during culture cycles. Low- and highly sulfated chondroitin sulfate, keratosulfate, and collagen, end products of differentiation in the cornea in vivo, were synthesized during both logarithmic and saturation phases of growth. Changes in the rate of sulfate incorporation into individual polysac-

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charides during the culture cycle did not parallel those of glucosamine incorporation, suggesting a change in the degree of sulfation of the polysaccharides synthesized as the cell density of cultures increased. Analysis of 68 clones of stromal fibroblasts, each doublelabeled with proline-3, 4-SH + NaZZ5S04 or proline-‘*C (uniformly labeled) + glucosamine-6-“Hi, showed that in direct proportion to their size 49 of these clones could make all three cornea1 polysaccharides and collagen. In all but one of the remaining smaller clones, all three polysaccharides were detected. The clonal analysis therefore suggested that each cell of this tissue-specific population of fibroblasts is specialized for the production of more than one end product of differentiation. ACKNOWLEDGMENTS

The unsparing assistance of Mrs. Linda S. Krulikowski is gratefully acknowledged. I wish to thank Dr. E. J. Boell for use of a liquid scintillation counter, Dr. F. H. Ruddle for use of a Coulter cell counter, and Dr. F. Glick for assistance with statistical analysis. I am indebted to investigators at the LaRabida-University of Chicago Institute, specifically Drs. A. Dorfman, A. C. Stoolmiller, J. A. Cifonelli, and M. B. Mathews, for helpful discussion and mucopolysaccharide standards. Dr. H. G. Coon contributed valued opinions on cloning techniques. The advice and encouragement of Dr. J. P. Trinkaus were especially appreciated. REFERENCES ABBOTT, J., and HOLTZER,H. (1966). The loss of phenotypic traits by differentiated cells. V. The effect of 5-bromodeoxyuridine on cloned chondrocytes. Proc. Natl. Acad. Sci. U.S. 59, 1144-1151.

ANSETH,A. (1961). Glycosaminoglycans in the developing cornea1 stroma. Exptl. Eye Res. 1, 116-121. BRAY, G. A. (1960). A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1, 279-285. BRINI, A., PORTE,A., and STOECKEL,M. E. (1966). Dheloppement de la cornbe chez l’embryon de poulet. Etude au microscope blectronique. Dot. Ophthalmol. 20, 309332.

CAHN,R. D. (1967). Detergents in membrane filters. Science 155, 195-196. CAHN, R. D., and CAHN, M. B. (1966). Heritability of cellular differentiation: clonal growth and expression of differentiation in retinal pigment cells in uitro. J+oc. Natl. Acad. Sci. U.S. 55, 106-114.

CAHN,R. D., COON,H. G., and CAHN, M. B. (1967). Cell culture and cloning techniques. In “Methods in Developmental Biology” (F. H. Wilt and N. K. Wessells, eds.), pp. 493-530. Crowell, New York. COLEMAN,J. R., HERRMANN,H., and BESS,B. (1965). Biosynthesis of collagen and noncollagen protein during development of the chick cornea. J. Cell Biol. 25, 69-78. CONRAD,G. (1970). Collagen and mucopolysaccharide biosynthesis in the developing chick cornea. Develop. Biol. 21, 292-317.

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COON,H. G. (1966). Clonal stability and phenotypic expression of chick cartilage cells in vitro. Roe. Natl. Acad. Sci. U.S. 55, 66-73. COULOMBRE, A. J. (1964). Problems in cornea1 morphogenesis. In “Advances in Morphogenesis” (M. Abercrombie and J. Brachet, eds.), Vol. 4, pp. 81-169. Academic Press, New York. DAVIDSON, E. H. (1964). Gene activity in differentiated cells. In “Retention of F’unctional Differentiation in Cultured Cells” (V. Defendi, ed.); Wistar Inst. Symp. Monograph 1, pp. 49-62. Wistar Inst. Press, Philadelphia, Pennsylvania. DAVIES,L. M., and PRIEST, R. E. (1967). Collagen synthesis in cultured cells: relations between collagen synthesis and cell proliferation. Fedemtion Z+oc. 26, 306 (Abstract No. 249). FRANKS, D., and DAWSON, A. (1966). Variation in the expression of blood group antigen A in clonal cultures of rabbit cells. Exptl. Cell Res. 42, 543-561. GOLDBERG, B., and GREEN,H. (1967). Collagen synthesis on polyribosomes of cultured mammalian fibroblasts. J. Mol. Biol. 26, l-18. GOLDBERG, B., and GREEN,H. (1968). The synthesis of collagen and protocollagen hydroxylase by fibroblastic and nonfibroblastic cell lines. Z+oc. Natl. Acad. Sci. U.S. 59, 1110-1115. GREEN, H., and HAMERMAN, D. (1964). Production of hyaluronate and collagen by fibroblast clones in culture. Nature 201, 710. HAM, R. G. (1963). An improved nutrient solution for diploid Chinese hamster and human cell lines. Erptl. Cell Res. 29, 515-526. HAMERMAN, D., TODARO, G. J., and GREEN,H. (1965). The production of hyaluronate by spontaneously established cell lines and viral transformed lines of fibroblastic origin. Biochim. Biophys. Acta 101, 343-351. HAUSCHKA, S. D., and KONIGSBERG, I. R. (1966). The influence of collagen on the development of muscle clones. Proc. Natl. Acad. Sci. U.S. 55, 119-126. HAY, E. D., and Rxvrr~, J.-P. (1969). Fine structure of the developing avian cornea. In “Monographs in Developmental Biology” (A. Wolsky and P. S. Chen, eds.), pp. l144. Karger, Basel, Switzerland. JAKUS, M. A. (1956). Studies on the cornea. II. The fine structure of Descemet’s membrane. J. Biophys. Biochem. Cytol. 2, Suppl. 4, 243-252. KONIGSBERG, I. R. (1963). Clonal analysis of myogenesis. Science 140, 1273-1284. LEVINE, E. M., BECKER, Y., BOONE, C. W., and EAGLE, H. (1965). Contact inhibition, macromolecular synthesis, and polyribceomee in cultured human diploid fibroblasta. hoc. Natl. Acad. Sci. U.S. 52, 350-356. LLOYD, A. G. (1959). Studies on sulphatases. The use of barium chloranilate in the determination of ensymatically liberated sulfate. Biochem. J. 72, 133-136. LUKENS, L. N. (1965). Evidence for the nature of the precursor that is hydroxylated during the biceynthesis of collagen hydroxyproline. J. Biol. Chem. 240,1661-1669. LUKENS, L. N. (1966). The size of the polypeptide precursor of collagen hydroxyproline. Z4oc. Natl. Acad. Sci. U.S. 55, 1235-1243. MATHEWS, M. B., and INOIJYE, M. (1961). The determination of chondroitin sulfate Ctype polysaccharides in mixtures of other acid mucopolysaccharides. Biochim. Biophys. Acta 53, 569-513. MEYER, K., DAYIDSON, E., LINKER, A., and HOFFMAN, P. (1956). The acid mucopolysaccharides of connective tissue. Biochim. Biophys. Actu 21, 566-518.

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