Isolation of phenotypically distinct trophoblast cell lines from normal rat chorioallantoic placentas

Isolation of Phenotypically Distinct Trophoblast Cell Lines from Normal Rat Chorioallantoic Placentase

JOAN S. HUNT”, SANTANU DEBb, TERESA N. FARIAby”, DAVID WHEATON”, & MICHAEL J. SOARES’,” Departments of Pathology” and Ph,ysiolo&, Ralph I,. Smith Mental Retardation Researrh Center, Universi(ll of Kansus Medical Center, Kansas C&y, Kansas 66103, USA Supported in part

b.y a Fulbright Predoctoral FeLlowship.

,’ To whom ail correspondrnce should be addressed.

r’Supported in part by grants jkom the National Institutes of Health Biomedical Research Program (BRSG SO7 RR05.373) and the March of Dimes Foundation for Birth Defects to 3.S.H. and by grants from the National Institutes of Child Health and Human Development, HD20676 and HD22208, and the Flossie West Memorial Trust to M.3.S. Paper accepted 2.3. I 1. I oXX

INTRODUCTION

The difficulties encountered by investigators in obtaining pure populations of trophoblast cells from placentas and in maintaining those cells in vitro have been major obstacles to the study of normal trophoblast cells (Rossant and Croy, 1985; Loke and Hussa, 1987). Primary cultures of trophoblast cells are frequently contaminated with maternal and fetal mesenchymal cells (Ferguson and Palm, 1976; Loke et al, 1986) and tend to differentiate rapidly into the end-stage giant cells (Rossant and Tamura-Lis, 1981; Soares and Glasser, 1987; Kliman et al, 1986; Kliman, Feinman and Strauss 1987). As a result, much of the information available regarding trophoblast cells has been obtained by studying their neoplastic counterparts, whose characteristics may or may not parallel those of normal trophoblast cells. In 1981, Log and co-workers reported a method for generating long-lived populations of cells from midgestation mouse placentas. The placental cells exhibited tumor-like characteristics in that cells proliferated rapidly in vitro and when transplanted in vivo. Mouse placental cells were not fully characterized and were used for a limited number of studies (Log, Chang and Hsu, 1981; Tanaka et al, 1983). By adopting the procedures described by the earlier investigators, we recently generated a long-lived cell line from midgestation placentas of outbred rats (Soares et al, 1987). The generation procedure did not include any manipulations known to effect transformation. The cells were uniformly epithelioid (Hunt et al, 1988) and exhibited patterns of antigen expression associated with trophoblast cells in situ (Hunt and Soares, 1988). When transplanted in vivo, the placental cells displayed an increased tendency to form or43--4004/89/020r6r + ‘7 $og.oo/o

c> 1989 Baillike Tindall Ltd

162

Plac-entu (1989), Vd. IO

giant cells and to express the trophoblast marker alkaline phosphatase (Soares et al, 1987). Placental cells were suceptible to growth regulation by exogenously supplied molecules: proliferation in vitro was enhanced by transferrin (De, Hunt and Soares, 1988) and suppressed by analogues of cyclic adenosine-3’,5’-monophosphate (CAMP) (Soares et al, in press). The extracellular matrix component laminin was identified as a major product of placenta1 cells maintained both in vitro and in vivo (Soares et al, 1988). Identification of the original cell line as trophoblast-derived was tentative because those cells had limited expression of some expected differentiation-associated trophoblast cell markers. In the present study, we report the generation of an additional five cell lines from outbred (Holtzman) and inbred (Lewis, PVG.RTrrB) rat placentas. All of the lines demonstrated a basic set of characteristics that are strongly associated and probably exclusive to trophoblast cells. In addition, each of the lines had individual features that may be useful in evaluating specific trophoblast cell structures and functions. Normal trophoblast cell lines with distinct phenotypes that are easily maintained in vitro and are susceptible to in vitro manipulation provide a valuable new approach for studying placental morphogenesis.

MATERIALS

AND METHODS

Generation and maintenance of trophoblast cell lines Cell lines were derived from midgestation chorioallantoic placentas of Holtzman x Holtzman (HRP.1, HRP.2a, HRP.5), Lewis x Lewis (LRP.2, LRP.8) and PVG.RTI~ x PVG.RTI’~ (R8RP.3) rats as previously described (Soares et al, 1987). Holtzman rats were obtained from the Holtzman Co. (Madison, WI) and Lewis rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN). PVG.RTI’~ rats were a generous gift from R. N. Smith, Case Western Reserve University. In brief, labyrinthine placentas obtained from day II or 12 of gestation were grown as explants in medium (RPM1 1640 containing antibiotics, 2 mM L-gtutamine, I mM sodium pyruvate and 50 PM z-mercaptoethanol) supplemented with 20 per cent fetal bovine serum (FBS) at 37”C, 5 per cent CO,. Outgrowths of explants were expanded and maintained in vitro in medium containing IO per cent FBS. In vitro passages 5-20 of the cell lines were used for this study. One of the six lines (HRP.1) received an in vivo passage by culture in the peritoneum of female Holtzman rats as described (Soares et al, 1987). Analysis of size distribution and DNA content by flow cytometry Cells were scraped from tissue culture flasks and dispersed by vigorous pipetting. Single cell suspensions were obtained by filtering cell suspensions through nylon mesh (pore size 74~ Small Parts, Miami, FL), and were adjusted to 5 x 10~ cells/ml in phosphate buffered saline (PBS, IO mM sodium phosphate, 0.9 per cent NaCl, pH 7.2). Cell size was analyzed by forward angle light scatter using an EPICS V (Coulter Electronics, Hialea, FL). For determinations of relative DNA content in cell nuclei, 0.5 ml of a hypotonic solution of prapidium iodide containing RNAse (0.005 per cent propidium iodide, Calbiochem-Behring, Sen Diego, CA; 0.02 per cent RNAse A, Sigma, St. Louis, MO; 0.3 per cent Nonidet P40, Sigma; 0.1 per cent sodium citrate) was added to I x 10~ cells as previously described (Krishan, 1975; McDivitt, Stone and Meyer, 1984). DNA content in isolated nuclei was analyzed by integral red fluorescence after 30 minutes incubation at 4% Morphology of cells by light microscopy Cells harvested by brief trypsinization from tissue culture flasks were seeded into chambers

Hunt rt 01: Trophoblart (5

x

Phemyptc

10~ cells/chamber

Tek Tissue

Culture

Vurrants

in

ml of medium

0.2

Chamber

with hematoxylin

microscope

equipped

each preparation each line.

Expression

as described

After

concentration

incubated

with a I: 30 dilution

of fluorescein

in PBS. A Zeiss fluorescence

relative

cell numbers

were estimated

rates of proliferation

stained

cells (Gillies,

mined

by incorporation

vested

from

tissue

Didier

and Denton,

of [ 3H]-methyl

culture

culture

tal violet

( I:I absolute

violet) was added

ethyl

were terminated

a1cohol:ro

The crystal

was eluted

medium

by adding

I 5 minutes

incubation

using a Titer-Tek

Multiskan

into 96 well flat 5 per cent

onto each plate, with

at days 1-7 following was removed

seeding.

Plates

and 0.05 ml of a solution formalin

were incubated period,

was removed

containing

microplates

low speed

reader (Flow Lab., MacLean,

for

were washed

in

The dye, which stains

ether (Sigma) shaking

(RT)

were again centri-

and the plates

glycol monomethyl

were

of crys-

0.5 g crystal

at room temperature

Plates were air dried overnight.

at RT with continuous microplate

from

was deter-

containing

all lines were seeded

of the incubation

0.2 ml of ethylene

activity

and were seeded

in 0.2 ml medium

per cent buffered

violet solution

tap water for 5 minutes.

of crystal violet eluted synthetic

For the crystal violet assay, cells were har-

trypsinization

per experiment;

the final 5 minutes

fuged at low speed. DNA,

thymidine.

to each well and the plates

During

gently running After

Plates

measurement

at IOO cells/well

at lou speed for 5 minutes,

I 5 minutes.

the slides.

1986), and (b) DNA

flasks by brief

microplates

of IO wells/line.

centrifuged

(Hialea,

using 80 per cent glycerol

of the cell lines, two types of assays were used: (a)

by spectrophotometric

FBS. Seven plates were established replicates

or an

then were

IgG from Coulter

with a coverslip

was used to examine

Finland)

Slides were washed,

of cell lines

To establish

tissue

microscope

Helsinki,

anti-mouse

and

to permeabilize

in PBS, then were incu-

Labsystems,

conjugated

and were mounted

into Lab-Tek

were disassembled

in cold acetone

mouse IgG for 30 minutes.

in

cells

Cells were rehydrated (PKKI,

using a

large cells (35-50 ,u) in

chambers

for 7 minutes

for

cells were evaluated

flasks were seeded

incubation

until tested.

of normal

FL). Slides were again washed,

bottom

48 hours

by light microscopy hundred

by trophoblast culture

I,ab-

were incubated

and the cell layers were

of unusually

filaments from tissue

of anti-cytokeratin

equivalent

rates

grid. Three

in PBS. Slides were immersed

with a I:ZO dilution

IL). Slides

were removed

percentages

intermediate

the cells and were stored at - 7oC

Growth

assemblies

the approximate

above.

5 per cent FBS) of g-chamber

Naperville,

Cell layers were evaluated

by brief trypsinization

slides were rinsed bated

Chamber

and eosin.

of cytokeratin

chambers

Lab.,

with an ocular measurement

to obtain

Cells harvested

containing

Slides (Miles

48 hours at 37°C in 5 per cent CO,. stained

‘63

to each well.

the plates

were read

VA) at a wavelength

of

520 nm. For the thymidine culture

incorporation

flasks were seeded

assay, cells harvested

into 96 well flat bottom

by brief trypsinization

microplates

at 2 x 10~ cells/well

from tissue in 0.2 ml

of medium containing 5 per cent FBS. Two microplates, each containing all 6 cell lines in replicates of 5, were established. Eight hours prior to the termination of the assay, I &i/well of [31~]-methy1

thymidine

(ICN

Radiochemicals,

Irvine,

CA) was added.

Plates were terminated

at 24 and 48 hours using a cell harvester (Skatron Co., Sterling, VA). Incorporated radiolabel in the filters was solubilized with ScintiVerse II (Fisher Scientific, Fair Lawn, NJ) and counted in a beta scintillation counter.

164

Regulation of DNA synthesis by transferrin The method used to evaluate the effects of transferrin on trophoblast cell DNA synthesis has been described previously (De, Hunt and Soares, 1988). The assay used in this study differed only in that assays were carried out using 96 well rather than 24 well flat bottom plates. Wells contained 2 x 10~ cells in 0.2 ml of medium, and tests were performed in replicates of 6. Expression of surface transferrin receptors The expression of surface transferrin receptors by trophoblast cell lines was evaluated by enzyme immunoassay. Cells harvested from tissue culture flasks by brief trypsinization were adjusted to 5 x 10~ cells/ml in medium containing IO per cent FBS, and 0.1 ml was added to each well of a 96 well flat bottom tissue culture plate. A single plate was used for each assay, with replicates of 7 wells per cell line. Plates were incubated at 37°C 5 per cent CO, for 4 hours to allow cell attachment, then plates were centrifuged at low speed for 5 minutes. Medium was removed and replaced with 0.05 per cent glutaraldehyde in PBS. After 20 minutes incubation at RT, the fixation reaction was terminated by removing the glutaraldehyde, adding 0.2M glycine in PBS to quench the reaction, and incubating at RT for 30 minutes. Plates were washed three times with 0.1 per cent BSA in PBS containing 0.02 per cent NaN,, were sealed with parafilm, and were stored at 4°C until tested. Plates were washed three times in o. I per cent BSA/PBS, then were incubated for 60 minutes at RT with o. I ml/well of either a mouse monoclonal antibody to the rat transferrin receptor (I : 800 dilution, approximately 0.1 pg/ml of MCA 155, Serotec, distributed by Bioproducts for Science, Inc., Indianapolis, IN) (3 wells), or an equivalent concentration of normal mouse IgG (3 wells) (Sigma). The seventh well received only buffer, and served as a control for nonspecific binding of the developing reagents. Plates were again washed three times, then o. I ml of a I : 500 dilution of goat anti-mouse IgG conjugated to horseradish peroxidase (Tago, Burlingame, CA) was added, and the plates were again incubated at RT for 60 minutes. After washing the plates three times, 0.1 ml of substrate [phosphate citrate buffer, pH 5.0, containing 0.012 per cent H,O, and 0.4 mg/ml o-phenylenediamine (Sigma)] was added. The plates were incubated for 30 minutes at RT, then absorbance was read at 492 nm using a Titer-Tek Multiskan microplate reader. Regulation of DNA synthesis by dibutyryl CAMP The procedure used to evaluate the ability of dibutyryl CAMP to suppress the incorporation of [3H]-methyl thymidine by trophoblast cells has been described (Soares et al, in press). For this study, the procedure was performed on 96 well microplates. In brief, cells were seeded at 2 x 10~ cells/well in medium containing 5 per cent FBS (5 replicates) or in the same medium to which I mM dibutyryl CAMP (Sigma) had been added (5 replicates). Assays were concluded after 24 hours incubation at 37°C 5 per cent CO,. Eight hours prior to conclusion of the assay, I &i/well [3H]-methyl thymidine was added. Incorporation of the radiolabel was determined as described above. Evaluation of conditioned medium for major extracellular products of trophoblast cell lines Cells harvested by brief trypsinization from tissue culture flaskswere seeded into 35 mm diameter plastic dishes (5 x 10~ cells in 1.0 ml medium containing IO per cent FBS), and were incubated at 37X, 5 per cent CO,, for 48 hours. Four hours prior to conclusion of the assay, the medium was replaced with I ml of methionine-free RPM1 1640 cutture medium (Sigma) containing [” 5S]-methionine (I IZO Ci/mmole, ICN Radiochemicals) at a concentration of IOO #Zi/ml. Following incubation, culture media were collected, nonradioactive L-methionine was

Hunt et ul: Trophoblost Phemqpzr

Vmants

16j

added to a final concentration of IO mM, and proteins were precipitated from the medium by the addition of cold acetone (z : I v). The precipitated proteins were collected by centrifugation, were solubilized in sodium dodecyl sulfate (SDS)-electrophoresis buffer (0.0625 M Tris-HCl, pH 6.8, 2 per cent SDS, IO per cent glycerol and 5 per cent 2-mercaptoethanol) and were heated at 90°C for 4 minutes. Samples were clarified by centrifugation and supernatants were electrophoretically separated in 7.5 per cent SDS-polyacrylamide gels (Laemmli, 1970). The gels were then stained with Coomassie Blue, were destained, and were then incubated sequentially in water for 30 minutes and I M sodium salicylate for 30 minutes. Gels were dried and exposed to Kodak X-OMAT AR X-ray film at - 7oC (Bonner, 1984). The presence of immunoreactive laminin in medium conditioned by each cell line was determined by an enzyme-linked immunoassay procedure similar to that previously described by Williams et al (1987). An antiserum to rat laminin was used at a final dilution of 1:45 ooo and purified rat laminin was used as a standard (Soares et al, 1988). Evaluation

of alkaline phosphatase

expression

by trophoblast

cell lines

Two approaches were used to evaluate alkaline phosphatase expression: (a) substrate conversion by populations of cells in microwells was measured spectrophotometrically, and (b) monolayers of cell were examined for bound converted substrate using light microscopy. For the first assay, cells harvested by brief trypsinization from tissue culture flasks were seeded into 96 well flat bottom microplates (5 x 10~ cells/well in 0.2 ml of medium containing 5 per cent FBS) with replicates of ten wells per cell line. Each plate contained all six cell lines. Plates were terminated at 24 and 48 hours by carefully removing the medium and adding 0.2 ml of substrate (z mg/ml disodium p-nitrophenyl phosphate from Sigma in buffer containing IOOmM Tris-HCl, pH 9.5, IOO mM NaCl, and 5 mM MgCl,). Plates were incubated for 15 minutes at RT and the reaction was terminated by adding 0.05 ml of 2M NaOH. Plates were read using a Titer-Tek Multiskan plate reader at a wavelength of 405 nm. For the second assay, cells were seeded into chambers of %Chamber Lab-Tek Tissue Culture Chamber/Slides at 2 x 10~ cells/well in 0.2 ml of medium containing 5 per cent FBS. After 48 hours incubation, the medium was removed, and substrate (0.05 mg/ml s-bromo-qchloroindoxyl phosphate and 0.1 mg/ml nitroblue tetrazolium, both from Sigma, in the Tris buffer described above) was added. After 15 minutes incubation at RT, the chambers were disassembled, slides were rinsed in PBS, and were counterstained lightly with methyl green. Slides were evaluated by light microscopy. Identification

of immunoreactive

placental

lactogen-II

(PL-II)

PI.-11 immunoreactivity in cell monolayers was identified by using a rabbit antibody to a synthetic peptide identical to amino acids 56-70 of rat PL-II (Deb et al, in press). Immunocytochemistry was performed on monolayers of two types of cells: trophoblast cells (all lines) and rat fetal fibroblasts. The latter cells were established from fetal limb buds obtained from Holtzman rats on day I 2 of gestation. Cells harvested by brief trypsinization were seeded into LabTek chambers at 5 x 10~ cells/well in 0.2 ml of medium containing IO per cent FBS, and were incubated for 48 hours at 37°C. Chamber assemblies were removed and the slides were air dried for 1-3 hours. Slides were immersed in cold acetone for 7 minutes and stored as before. Slides were rehydrated, then were incubated with dilutions (I: 200, 1:400 in PBS) of preimmune serum, immune rabbit serum or immune serum preincubated with IO pg/ml of the synthetic PL-II peptide. Antibody binding to monolayers of cells was identified using an antirabbit IgG avidin-biotin immunoperoxidase staining system from Vector Laboratories (Vectastain ,4BC, Burlingame, CA). Slides were lightly counterstained with Gill’s hematoxylin.

I66

RESULTS Morphology by light microscopy Figure I illustrates the similarities and differences among the six cell lines grown on glass slides and stained with hematoxylin and eosin. Cells in all lines had basophilic cytoplasm. Some cells in all of the lines contained eosinophilic inclusions that have been shown (in the HRP. I line) to contain laminin (Soares et al, 1988). Cells in lines generated from outbred rats (HRP.1, HRP.2a, HRP.5) had a greater tendency to form colonies with flattened polygonal cells (arrowheads) than did cells generated from inbred lines (LRP.2, LRP.8, RSRP.3). Extremely large cells with diameters of 35-50 p (arrows) were a common feature in 3 lines (HRP.2a, HRP.5, LRP.8; 3-5 per cent of the total cells) when cells were grown in medium containing 5 per cent FBS but were uncommon in the other 3 lines (
Figure I. Monolayers of six rat trophoblast cell lines grown on glass slides and stained with hematoxylin and eosin. (a) LRP.2, (b) HRP.1, (c) HRP.za, (d) LRP.8, (e) HRP.5, (f~ RSRP.3. Flattened polygonal cells are indicated with arrowheads, and cells with a diameter of 35-50 p are indicated with arrows: x 175.

ffnnt

ct irl: hphohlmt

LRP.z

or HRP.1

Phenqyw

‘67

cells) were grown in medium containing

large cells increased HRP.2a

C’orrrrnt.\

to 3-5 per cent.

Binucleate

line than in other lines. Multinucleated

cell lines except HRP.1 Size distribution Observation

and DNA content

the different

scatter

patterns

per cent FBS, the numbers

cells were occasionally

observed

observed

of

in the

in all of the

and LRP.2.

of cells in monolayer

among

10-20

cells were more frequently

analysis

culture

by flow cytometry

suggested

cell lines. That observation

that the distribution

was confirmed

of the lines using flow cytometry.

Figure

by analyzing

of cell sizes varied the forward

light

z shows that cells in two cell lines,

were, on the average, considerably smaller (lower mean channel) than cells in the other four lines. Sharper peaks in the same two cell lines indicated a greater uniformity of size than was characteristic of other cell lines. The mean channel for LRP.2 cells was 95 and

HRP. I and LRP.2,

for HRP.1 (I,RP.X),

cells was 99. Mean 149 (HRP.5)

When the nuclear staining

(Figure

(zn, channels

channels

for the other

content

of DNA for each cell line was determined

3), all cell lines were shown to contain

31~ 34) and tetraploid

(4n, channels

LRP.2

HRP.20

(c)

I

BL

(e)

four lines

were

161 (HRP.2a),

165

and 161 (R8RP.3).

,

using propidium

the DNA levels associated

iodide

with diploid

62-6X) nuclei. The DNA content

of tropho-

(b)

HRP.1

(d)

LRP.8

I

80

GL

HRP.5

Chonnel

R8RP.3

Number

FI~UW 2. Size distribution analysis of cells in six trophoblast cell lines by forward angle light scatter in flow cytometry. To obtain the size of the median cell in each line, debris in the lower channels (< 62) were eliminated, and analy-siswas performed ofgates 62- 256. (a) LRP.2, (b) HRP.,, (c) HRP.za, (d) LRP.X, (e) HRP.;, (r) RSRP.3.

168

Placentu (1989), Vol. 10

0

31

66

I26

LRR 2

(b)

HRP.1

HRP. 20

(d)

LRP.6

HRP.S

(f )

256

0

RBRP.3

31

66

126

256

Chonncl Number Figure 3. Nuclear DNA content of cells in six cell lines determined

cytometry. The control cell population was rat thymocytes, stainless steel mesh. The sharp zn peak for thymocytes (c) HRP.za, (d) LRP.8, (e) HRP.5, (r) R8RP.3.

by propidium iodide staining with analysis by flow which were obtained by screening minced thymus through is shown in black (channel 31). (a) LRP.2, (b) HRP.1,

blast cell nuclei in the diploid peaks was invariably higher than that of rat thymocyte nuclei used for comparison. One cell line, HRP.5, contained nuclei with fin content (channel IZ~), suggesting giant cell formation. Intermediate filament expression by trophoblast eel1 lines When cells grown on slides were stained by indirect immunofluorescence to identify cytokeratin intermediate filaments, the cells in all six lines were positive. In smaller size cells, filaments were usually concentrated near the nucleus whereas linear arrays of filaments were observed in larger size cells (not shown). Control slides of cells incubated with an equivalent concentration of normal mouse IgG as a substitute for the anti-cytokeratin mouse monoclonal antibody were negative. Growth characteristics The rates of growth of in vitro cultured trophoblast cell lines were compared by seeding cells at low density, then identifying the numbers of cells on the microplates at days 17 using the crystal violet assay. Two cell lines (LRP.2 and HRP.1) grew rapidly in medium containing 5

Hunt rt al: Trophnblast Phemyptc

Varimts

169

I

0

2

4

3

5

7

6

Ooy of culture Figure 4. Growth of six trophoblast cell lines over a seven day time period. Cells were plated at low density in medium containing 5 per cent FBS, and plates were terminated at days 1-7. The cell densities over time were estimated by spectrophotometric measurement (absorbance, 520 nm) of crystal violet eluted from crystal violet-stained cells. Points on the figure represent the mean and standard error of the mean for IO replicates.

per cent FBS whereas cation (Figure cent FBS). ive rates

the HRPza,

4). The RSRP.3

Cells growing of DNA

in medium

synthesis

8 hours and determining obtained

LRP.8,

and HRP.5

lines had more moderate

line grew very slowly under containing

rates of repli-

of the assay (5 per

5 per cent FBS were also tested for comparat-

at 24, 48 and 72 hours

the amount

the conditions

of incorporated

by adding

[3H]-methyl

thymidine

label. The same comparative

for

results were

(not shown).

Both fast and slow growing concentrations

cell lines responded

of FBS (not shown).

ing lines (LRP.2,

HRP.

cent FBS whereas

I)

Additional

could be maintained

slower growing

with increased

experiments

with ease in medium

lines (LRP.8,

HRP.5,

proliferation

demonstrated RSRP.3)

to increasing

that the fast grow-

containing required

as little as a minimum

2

per

of IO

per cent FBS for maintenance.

Responses Figure

to transferrin

3 shows

and expression

that the addition

of surface

of rat transferrin

transferrin

to cultures

receptors

of cells growing

in serum-free

medium stimulated the incorporation of [3H]-thymidine by all six cell lines. The cell lines varied in their responsiveness to transferrin as a growth supplement. Receptors for transferrin were expressed

by cells in all lines. Absorbance

values ranged

from 0.242 to 0.489 for the six

lines.

Responses

of trophoblast

cell lines

to exogenous

dibutyryl

CAMP

Figure 6 shows that incorporation of r3H]-methyl thymidine by five of the six cell lines was inhibited by the addition of dibutyryl CAMP to cell cultures. A direct relationship between the rate of cell proliferation

and the ability of CAMP to inhibit

proliferation

was suggested

by the

results. In both of the rapidly proliferating cell lines, LRP.2 and HRP.1, incorporation of the radiolabel was inhibited by 68 per cent. Cell lines that grew at a more moderate rate (HRP.za, LRP.8,

HRP.5)

were inhibited

by 37-48 per cent whereas

the R8RP.3

line, a very slow grower

2

??Control ? ?Tronsferrln

80

2

0 LRP.2

HAP. I

HRP.20 LRP. 8 Cell lines

HRP.5

RBRP. 3

Figures. Stimulation of DNA synthesis by transferrin. Trophoblast cells were plated in medium containing 5 per cent FBS for 24 hours, then medium was replaced with serum-free medium and tlte cells were grown for an additional 24 hours. Serum-free medium was replaced with either serum-free medium (closed bars) or with serum-free medium containing 5 ,ug/ml of rat transferrin (hatched bars), and the cells were incubated for an additional 16 h. I Ki/well of [SH]-thymidine was added and the plates were terminated 8 hours later. The means and standard errors of the means are shown for replicates of six wells.

sor

II

Control

m

Dibutyryl

CAMP

Cell lines

Figure 6. Inhibition of DNA synthesis by dibutyryl CAMP. Trophoblast cell lines were grown in microplates in medium containing 5 per cent FBS without (closed bars) or with (hatched bars) I .o mM dibutyryl CAMP. DNA synthesis was identified by the incorporation of [3H]-methyl tbymidinc into the cells. The means and standard errors of the means are shown.

Hunt et al: Trophoblast Phenotypic Variants

‘71

(cl

(d)

(e)

If

1 _ominin A

200 Kd

_ammln B

I16 Kd

97 Kd

66 ) Kd

-

iKd

Fzgure 7, Major extracellular products of rat trophoblast cell lines. After a 4 hour incubation with [35S]-methionine, proteins in conditioned medium from cell cultures were separated by electrophoresis on a 7.5 per cent polyacrylamide gel in the presence of SDS. The gels were evaluated by fluorography. Lane (a), LRP.2; lane (b), LRP.8; lane (c), R8RP.3; lane (d), HRP.J; lane (e), HRP.aa; lane(f), HRP.5. Approximate molecular weights of the protein bands are indicated to the left, and some distinctive bands on the right.

in medium containing ment (20 per cent).

5 per cent FBS, demonstrated

slight but not significant growth enhance-

Extracellular products of trophoblast cell lines The extracellular products of the cell lines were identified by adding [35S]-methionine to cells growing in methionine-deficient medium and harvesting the conditioned medium after 4 hours. The radiolabelled products were evaluated by polyacrylamide gel electrophoresis followed by fluorography (Figure 7). The major bands were similar but were not identical in the six cell lines. All lines contained proteins migrating as expected for the A and B chains of laminin (Soares et al, 1988). The presence of laminin in conditioned medium from each cell line

Phcentu (1989), Vol. IO

1.0

0.5

0.0 Cell

Figure 8. Expression of alkaline phosphatase by trophoblast sion in a microplate assay. Bars indicate mean absorbance mean are shown.

lines

cells at 24 and 48 hours as determined by substrate convervalues at 405 nm for replicates of IO wells, and se. of the

was confirmed by enzyme immunoassay (not shown). Conspicuous differences among the lines were observed in proteins migrating in the 150 Kd and 43 Kd regions (arrows). The 150 Kd protein was synthesized by the slower-growing cell lines (LRP.8, HRP.5, R8RP.3), but not in detectable amounts by the three fastest-growing cell lines (LRP.2, HRP.1, HRP.za). Only RBRP.3, the slowest-growing line, failed to synthesize detectable levels of the 43 Kd protein. Expression of alkaline phosphatase Figure 8 demonstrates that trophoblast cells obtained by brief trypsinization then grown for 24 hours in microwells exhibited low levels of expression of the enzyme alkaline phosphatase as evaluated by the ability to convert substrate. After 48 hours in culture, the expression of alkaline phosphatase increased in all lines. The highest levels of substrate conversion were associated with moderate rates of growth; lower levels were associated with cell lines that grew both rapidly (LRP.2, HRP.1) and slowly (RSRP.3). Histochemical identification of alkaline phosphatase positive cells within each cell line was also performed. Cells from each line were grown on slides, substrate was added, and the monolayers were examined for substrate conversion (Figure 9). Observation by light microscopy confirmed relatively poor substrate conversion by fast growing lines FRP.2, Figure 9(a)]. Cell

Hunt et al: Trophoblast Phenot,ypic Variants

‘73

Frgure 9. Deposition of alkaline phosphatase substrate by two of the six trophoblast cell lines. (a) LRP.2 cells are lightly stained. (b) LRP.8 cells demonstrate more intense staining. High levels of substrate deposition were confined to specific cells): x ,000

lines with high values in the first type of assay exhibited higher levels of substrate conversion [LRPJ, Figure 9(b)]. High expression was limited to specific cells within each line, being most prominent in aggregates of cells. Immunoreactive placental lactogen-II (PL-II) in trophoblast cell lines Immunocytochemical staining of cell layers with a rabbit antiserum generated to a synthetic peptide corresponding to amino acids 56-70 of rat PL-II (Deb et al, in press) demonstrated that the antiserum bound to nearly all cells in all six trophoblast cell lines. Some cells in all lines were stained more strongly than others. LRP.2 cells demonstrated weak binding of the antiserum in comparison with the other cell lines. Preimmune serum did not bind to trophoblast cell monolayers [Figure IO(a)], and binding of immune serum was completely inhibited by preincubating the immune serum with IO pg/ml of the immunizing peptide [Figure IO(e)]. Monolayers of fetal fibroblast cells did not bind preimmune, immune, or peptide-inhibited antiserum [Figures Io(b,d,f)]. Summary In Table I the variable characteristics of the six cell lines are grouped so as to illustrate the general relationships that were demonstrated. A direct relationship was tentatively established among rate of growth of the line, cell line sensitivity to CAMP, and synthesis of a 43 Kd protein. An inverse relationship between those characteristics and serum dependency, expression

Placenta (1989), Vol.10

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(b)

Figure IO. Immunocytochemical staining of monolayers of cells with a rabbit antiserum generated to a synthetic peptide corresponding to amino acids 56-70 of rat placental lactogen-II (Deb et al, in press). Preimmune and peptideblocked anti-PL-II failed to bind to trophoblast cell monolayers, and fetal fibroblasts did not stain with anti-PL-II. (a, c, e) HRP.5. (b, d, f) Fetal fibroblast cells. (a, b) Preimmune serum at a 1:400 dilution. (c, d) Anti-peptide antiserum at a r:400 dilution. (e, f) Anti-peptide antiserum (1:400) preincubated with IO pg/ml of the immunizing peptide. Binding of the antiserum was detected with an avidin-biotin immunoperoxidase staining system (anti-rabbit IgG ABC kit from Vector Lab.). Slides were lightly counterstained with Gill’s hematoxyline: x 700.

of the I 50 Kd protein and containment of immunoreactive PL-II appeared to be present. High levels of dependency on transferrin for DNA synthesis and alkaline phosphatase activity were generally associated with moderate rates of growth.

DISCUSSION Morphogenesis of the chorioallantoic placenta requires appropriate orchestration of interactions among trophoblast, fetal, and maternal cells. The availability of pure populations of the interacting cell types would greatly facilitate dissection of those interactions. In this report we describe the growth and differentiation-associated characteristics of six placenta-derived cell lines of trophoblast lineage.

Hunt et ul: 7iophoblart Phenoyptc Varrants

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Table I. Summary of trophoblast

Characteristic

LRP.2

cell’line characteristics,’

HRP.1

HRP.2a

Cell line LRP.8

HRP.5

R8RP.3

Mean Cell Six

Small

Small

Large

Large

Large

I.arge

Rate of Growth CAMP Sensitivity 43 Kd Protein

Fast High Yes

Fast High Yes

Moderate Moderate Yes

Slow LOW Yes

Slow Low Yes

Slow None No

Serum Dependency rso Kd Protein PI.-11

LOW No LOW

ND” No High

ND No High

ND Yes High

ND Yes High

High Yes High

Transferrin Dependency Alkaline Phosphatase

Low

Moderate I.ow

High High

High High

Moderate Moderate

Low Low

LOW

“For comparisons, the relative values obtained in experiments performed simultaneously on the six trophoblast cell lines (Results and Figures 2,~,5,6,7,9, I I) are grouped into small and large, fast, moderate, and slow, high, moderate, and low. Identification of specific protein bands on polyacrylamide gels is shown as yes or no. fiND, not determined.

In accordance with previous reports from our laboratory on the behaviour of one of these cell lines (Soares et al, 1987; Hunt and Soares, 1988; De, Hunt and Soares, 1988; Soares et al, in press), the five newly derived rat trophoblast cell lines expressed cytokeratin intermediate filaments, transferrin receptors and alkaline phosphatase, and were responsive to growth regulation by transferrin and CAMP (with the exception of the R8RP.3 line). All of the lines synthesized the extracellular matrix component laminin (Soares et al, 1988). The availability of a recently characterized antiserum to rat PL-II (Deb et al, 1989) permitted the identification of immunoreactive cells within each of the trophoblast cell lines. PL-II is synthesized by trophoblast giant cells (Soares, Julian and Glasser, 1985) and is produced within both the junctional and labyrinthine regions of the chorioallantoic placenta (Soares, 1987). The significance of this report lies not in documentation of the similarities of the six trophoblast cell lines but in identification of their differences. Phenotypically dissimilar cell lines, all of which display trophoblast cell characteristics, provide a valuable group of experimental tools for clarifying the cellular and biochemical mechanisms involved in the control of trophoblast cell growth and differentiation. Specific characteristics that were variable among the lines were (i) morphology, (ii) cell size, (iii) cellular DNA content (iv) rates of proliferation, (v) serum dependency, (vi) responsiveness to transferrin and CAMP, (vii) types of proteins synthesized and released, (viii) expression of alkaline phosphatase, and (iv) expression of PL-II immunoreactive proteins. Phenotypic patterns among the cell lines were relatively difficult to identify, and the numbers of lines established did not allow statistical correlations to be drawn. However, some general associations were noted (Table I). Mean cell size and rate of growth seemed strongly associated, and the combination seemed to influence some other cell line characteristics. Of particular interest, the three slower-growing lines synthesized and secreted a ISO Kd protein that was not detectable in the medium conditioned by the faster growing lines. Identification of the 150 Kd protein, which may be similar to the laminin-binding extracellular matrix component entactin/nidogen (see review by Timpl et al, 1987), is the subject of current investigation. The slowest-growing cell line, RSRP.3, displayed a relatively unique secretory pattern of newly synthesized proteins; a 43 Kd protein synthesized by all of the other cell lines was absent. Compelling questions arise as a result of this study. Why, with essentially identical methods

Placenta (1989), v-4. 10

176

used for generating the trophoblast cell lines, do phenotypically dissimilar lines result? How does each of the lines relate to in vivo populations of trophoblast cells? Presumably, a trophoblast-derived stem cell population exists within the placenta that is capable of differentiating into phenotypically distinct subpopulations (Gardner, 1983; Rossant, 1986; Soares et al, 1987). The stages of differentiation toward the endstage endoreduplicated or multinucleated cell have not been identified. It seems reasonable to postulate that trophoblast cell differentiation is a multistage process, with numerous stable intermediates. Thus, the phenotypically disparate cell lines may represent trophoblast cells in various stages of differentiation that were selected at random during the generation of the lines. Since primitive types of cells are noted for their high rates of proliferation and low dependency on extrinsic factors for growth, the fast-growing lines more nearly resemble stem cells than differentiated cells. Slow-growing lines may have received differentiation signals in vivo. An important caveat, however, is that specific characteristics may have been altered by removing cells from their normal placental environment. In summary, we have isolated a group of trophoblast cell lines with distinct and disparate phenotypic characteristics. The value of the lines lies in their potential use as unique experimental models for studying the regulatory mechanisms involved in placental morphogenesis.

SUMMARY Growth characteristics and the expression of trophoblast-associated markers by six cell lines generated from midgestation chorioallantoic placentas of outbred (Holtzman) and inbred (Lewis, PVG.RTI”) rats were evaluated. The cells comprising all cell lines were epithelioid (contained cytokeratin-type intermediate filaments), had normal (271, 4n) DNA content, and synthesized the extracellular matrix glycoprotein laminin. Variability was observed among the lines in all other characteristics: median cell size, rate of growth, serum dependency, responses to transferrin and dibutyryl cyclic adenosine-3’,5’-monophosphate, synthesis of some major proteins, alkaline phosphatase activity, and the expression of immunoreactive placental lactogen-II. In general, cell lines with smaller mean cell sizes grew rapidly and required little serum for maintenance in vitro; cell lines with larger mean sizes grew more slowly and preferred higher concentrations of serum. Some associations between mean cell size/rate of growth and other characteristics were observed. No major differences were apparent between cell lines generated from outbred and inbred rat placentas. Trophoblast cell lines expressing distinct phenotypes provide a valuable new approach for studying a wide range of trophoblast cell activities.

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