Molecular and Cellular Endocrinology 228 (2004) 103–119

Review
Endocrine cell lines from the placenta
M.H.F. Sullivan∗
Department of Obstetrics and Gynaecology, Faculty of Medicine, Wolfson and Weston Research Centre for Family Health, Institute of Reproductive and
Developmental Biology, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK

Received 16 January 2003; accepted 31 March 2003

Abstract

Cell-lines derived from human placenta and chorion have been used extensively to model the endocrine functions of human trophoblast. In
general terms, the endocrine functions of the primary cells and tissues are at least partially replicated within the cell-lines, suggesting that they
may be used as appropriate models. There are, however, two major provisos that compromise this generalisation. Firstly, the endocrine function
of placenta represents a complex interaction between cytotrophoblast, syncytiotrophoblast and multiple regulators, so a single cell population
digested from the normal environment is unlikely to represent this. Secondly, the characterisation of primary trophoblast populations and of
cell-lines is incomplete, complicating the assignment of functions to trophoblast populations. Despite these difficulties, useful information
has been obtained from the available cell-lines, regardless of whether they have arisen spontaneously, been transformed in vitro, or derived
from cancers in vivo.
© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Endocrine cell lines; Placenta; Pregnancy

1. Defining the endocrinology of normal human
placenta
The placenta has a very wide range of functions in preg-
nancy, such that it has been described as an experimental
system in its own right, independently of its interactions
with fetal and maternal physiology. In seeking to identify
the endocrine functions of the human placenta, it is nec-
essary to consider what might be an appropriate defini-
tion. Classically, endocrinology has been defined as “The
study of the system of glands and tissues that produce and
secrete hormones”—the latter being active regulatory sub-
stances formed in one part of the body and carried by the
blood to another, where they signal the coordination of cel-
lular functions. Placental function does not necessarily fit
within these definitions, as placental products may have in-
trauterine targets as well as extrauterine targets, and only
the latter would necessarily include the blood-borne aspect.
In this review I propose to include all factors that are con-
sidered to part of the hypothalamic-pituitary-adrenal (HPA)
axis or the hypothalamic-pituitary-gonadal (HPG) axis in the
∗ Tel.: +44 20 7594 2133; fax: +44 20 7594 2154.
E-mail address: mark.sullivan@imperial.ac.uk (M.H.F. Sullivan).
non-pregnant adult. On this basis factors that have a regula-
tory role in placental endocrine function, but are not present
at increased levels in maternal blood during pregnancy may
be included in this review. For a fuller treatment of the HPA
and HPG axes, readers should refer to the appropriate chap-
ters elsewhere in this special issue of Molecular and Cellu-
lar Endocrinology, as well as general reviews.
The human placenta does not generally seem to be de-
pendent on these maternal endocrine systems, although it
can regulate the maternal HPA and HPG axes, and there
is cross-talk between the placental and maternal systems
(Glinoer et al., 1990; Lockwood et al., 1996; Petraglia et al.,
1996, 1998; Miller, 1998). It has the capacity to produce
factors that typify hypothalamic, pituitary and ovarian func-
tions, and the primary placental sources are listed in Table 1;
it must be pointed out that some of these factors are in-
cluded as regulators of placental function, and hence of the
outcome of pregnancy, rather than being part of the HPA or
HPG axes. These will be considered only in so far as they
contribute to the regulation of endocrine placental functions.
A series of papers and reviews have considered the nature of
placental endocrinology and its control (Albrecht and Pepe,
1990; Evain-Brion et al., 1990; Masuhiro et al., 1991; Shi
and Zhuang, 1993a,b; Stephanou and Handwerger, 1994;
Petraglia et al., 1995, 1996, 1998; Morrish et al., 1998; Reis

0303-7207/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
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104 M.H.F. Sullivan / Molecular and Cellular Endocrinology 228 (2004) 103–119

Table 1
Endocrine factors, endocrine regulators, and their production by the placenta
Hypothalamic products
GnRH VCT, SYNT Increased in maternal blood (Sorem et al., 1996)
CRH SYNT, VCT, CHOR Increased in maternal blood (Sorem et al., 1996)
Somatostatin VCT Increased in maternal blood (Goldstein et al., 1995)
GHRH No change in pregnancy (Mazlan et al., 1990)
Ghrelin VCT (Gualillo et al., 2001) Decreased in maternal blood (Makino et al., 2002)
TRH SYNT (Bajoria and Babawale, 1998) Increased in maternal blood (Amino et al., 1981)
Dopamine Decreased in maternal blood (Wang et al., 1999)
NPY VCT, CHOR Increased in maternal blood (Petraglia et al., 1989, 1993)
Pituitary products and homologues
Chorionic gonadatrophin SYNT Increased in maternal blood
Placental lactogen SYNT Increased in maternal blood
ACTH SYNT, CHOR Increased in maternal blood (Goland et al., 1992)
␤-Endorphin SYNT, CHOR Increased in maternal blood
␣-MSH SYNT, CHOR No change
Oxytocin SYNT, CHOR Increased in maternal blood
GH SYNT Increased in maternal blood (Mazlan et al., 1990)
Variant GH SYNT (Scippo et al., 1993) Increased in maternal blood (Eriksson, 1989; Frankenne et al., 1987, 1988)
Adrenal products
Glucocorticoids
Deoxycorticosterone Not produced Increased in maternal blood (Dorr et al., 1989; Goland et al., 1992)
Cortisol Not produced Increased in maternal blood (Lockwood et al., 1996)
Mineralocorticoids
Aldosterone Not produced Increased in maternal blood (Dorr et al., 1989)
Gonadal products
Progesterone SYNT, VCT, CHOR Increased in maternal blood
Estrogen SYNT Increased in maternal blood
Activins SYNT, VCT, CHOR Increased in maternal blood
Inhibins SYNT, VCT, CHOR Increased in maternal blood (Qu and Thomas, 1998)
Other local regulators
Interleukins CHOR Increased in maternal blood
Nitric oxide SYNT Increased in maternal blood (Shaamash et al., 2001)
TGF-␤ family SYNT
EGF SYNT, VCT
IGF-I, IGF-II SYNT, VCT, CHOR Increased in maternal blood (IGF-I)
IGFBP-1 Decidua IGFBP-1 increased in maternal blood
Colony-stimulating factor-1 Increased in maternal blood (late) (Tsakonas et al., 1995)
Fibroblast growth factors CYT
CRH-BP SYNT, VCT, CHOR Increased in maternal blood
Extracellular matrix Sanyal and Das (1997)
Hypoxia–HIF-1
The primary source of information is Petraglia et al. (1996), and references therein. Other papers are given to delineate specific data. No localisation
information is shown if this information is not available.

and Petraglia, 2001; Reis et al., 2001; Lacroix et al., 2002;
Schneider-Kolsky et al., 2002); the initial part of this review
will cover those aspects that are particularly pertinent to un-
derstanding the data described later from cell lines. In ad-
dition, thyroid hormones may amplify placental endocrine
activity in early pregnancy (Maruo et al., 1991), indicat-
ing that further regulatory pathways may modulate placental
function.
2. The localisation of placental endocrine functions
The primary complication in considering placental en-
docrinology is that different placental cell-types have dif-
ferent functions (Dockery et al., 2000). This differentiation
of function is outlined in Fig. 1, showing that the villous
cytotrophoblast are the main source of regulatory factors,
whereas the syncytiotrophoblast produce the “classic” pep-
tide and steroid hormones (Gaspard et al., 1980; Morrish
and Marusyk, 1997). This is not to say that cytotrophoblast
cannot produce hCG, hPL or steroids in vitro—many stud-
ies have shown that they can do so—but rather to emphasise
that the main physiological source is the syncytium of intact
placental villi. The functional differentiation must be consid-
ered in all in vitro studies of placental endocrinology. Any
separation of cytotrophoblast from the overlying syncytium
will immediately affect the functions of the syncytium, as
the normal regulation by cytotrophoblast products will be
removed. Furthermore there may be regulation in the op-
posing direction—some studies indicate that chorionic
 M.H.F. Sullivan / Molecular and Cellular Endocrinology 228 (2004) 103–119
Fig. 1. Cellular sources of the main endocrine factors of human pregnancy in (a) placenta and (b) fetal membranes.
gonadotrophin (syncytial product) regulates progesterone
production (a syncytial and cytotrophoblast function)
(Bhattacharyya et al., 1992; Chaudhary et al., 1992); again,
this will be lost on tissue digestion. This type of experimen-
tal separation is obligatory if the mechanisms controlling
hormone production are to be investigated with precision.
The localisation of mRNAs or proteins involved in the con-
trol of placental endocrine functions has been performed
in cultured explants or intact tissue samples, and of course
provides substantial information on the cellular sources, but
cannot easily be used to investigate intracellular mecha-
nisms and signal transduction pathways. A complication of
differential functions is that factors implicated in the con-
trol of placental endocrinology (see Table 1) may primarily
influence trophoblast differentiation, which in turn leads to
changes in endocrinology. For example, factors that increase
the formation of syncytia (e.g. EGF) (Morrish et al., 1987,
1997) will secondarily increase hCG and hPL production,
as these peptide hormones are produced at higher levels by
105
differentiated cells; however, some data indicate that differ-
ent factors may also exert specific regulatory effects on hor-
mone production (Feinman et al., 1986; Ritvos et al., 1988).
With these clear differences in cell biochemistry, it is nec-
essary to have reliable means of identifying the different pla-
cental cell populations, and this must be considered before
placental pathways and cell functions can be addressed.
3. Characterisation of different placental cell-types
The digestion of the human placenta produced a range of
cell-types (Aboagye-Mathiesen et al., 1996a). Gentle tech-
niques can be used to obtain preparations that are composed
mostly of syncytial trophoblast structures, which can then
be cultured. More vigorous techniques produce mixed cell
populations, which include villous cytotrophoblast, mononu-
clear syncytial fragments (which may superficially resemble
cytotrophoblast cells) (Huppertz et al., 1999), stromal and
106 M.H.F. Sullivan / Molecular and Cellular Endocrinology 228 (2004) 103–119
endothelial cells. This applies equally to first trimester and
to term preparations, as well as to chorion cells from fetal
membranes, which may be contaminated with decidual or
amnion cells. Extravillous trophoblast may also be obtained
from first trimester tissues (Tarrade et al., 2001).
Purified cells must be used if the data is to inspire confi-
dence that the data truly pertain to trophoblast, but this may
further remove the cells from their in vivo function which
may be controlled by the surrounding cells and matrix. Pla-
cental cell preparations may be crudely assessed by con-
firming the output of expected hormones (e.g. progesterone,
hCG), but more specific markers are needed to purify tro-
phoblast cells. Cytokeratin-7 seems to be expressed by all
trophoblast, and few other cells within the pregnant uterus
(Potgens et al., 2001). This is an intracellular protein, so it
is not of use in selecting viable cells, but it seems to be a
valid way to assess the overall purity of cell preparations.
The selection of viable cells has been considered in a
number of studies and summarised in Workshop Reports
(Frank et al., 2000a, 2001; Guilbert et al., 2002; Morrish
et al., 2002). The overall conclusion is that villous cytotro-
phoblast do not express any HLA class-I markers (Frank et
al., 2000a, 2001), so these cells can be purified by removal
of all other cell-types by pan-HLA antibodies. Such cells
are also CD9-negative, so depletion of CD9+ cells may also
purify villous trophoblast (Yui et al., 1994). The main con-
clusion is that careful characterisation of primary cell prepa-
rations is desirable if reliable data are to be obtained. This
is critical in the production of cell lines from primary cells,
as mixed cell starting populations may give rise to heteroge-
neous cell lines, and heterogeneous data. Gestational age will
also have an impact on the data obtained, as first trimester
cytotrophoblast do not form syncytia in vitro, whereas third
trimester cytotrophoblast do, and the reasons for this dif-
ference in differentiation of these apparently similar villous
cells are not understood.
Villous trophoblast are not the only possible type of
cells that may be appropriate for endocrine studies. There
are many different cell-types within the trophectoderm-
trophoblast lineage, but it seems likely that only two of these
are likely to have a major impact throughout pregnancy as
they are present in significant numbers. Extravillous cells
within the placental bed and anchoring villi are CK7 posi-
tive, but also express HLAG and CD9, which can be used
for definitive selection (Tarrade et al., 2001; Hirano et al.,
1999a). These cells are in immediate contact with maternal
cells, and may therefore play a critical role in regulating
the feto-maternal interface, and preparations of this type
have been used to study aspects of trophoblast motility or
invasion.
The chorion is also CK7 positive (Fig. 1b) and HLAG
positive (Hutter et al., 1996), and can synthesise a range of
endocrine products including progesterone, CRH and hCG
(Tonkowicz and Poisner, 1985; Jones et al., 1989; Cooper
et al., 1994). It is in immediate proximity to the maternal
decidua, and close to the cervix and myometrium. The fetal
membranes provide a larger area of interface with the mater-
nal tissues than does the placenta; as cervix and myometrium
are integral to the process of parturition, the regulation of
their activity by chorionic products may be important in the
continuation of human pregnancy.
4. The endocrinology of primary cells from the human
placenta
It is necessary to review the information obtained from
isolated human primary placental and trophoblast cell prepa-
rations, so that the data from cell lines can be placed in the
correct context. These primary cultured cells, and villous
explants, have been used to generate a large body of data on
the regulation of placental endocrine functions. Primary tro-
phoblast cells have generally been prepared from two major
sources, namely first trimester termination tissue, and term
placenta or fetal membranes after delivery by elective Cae-
sarean section or after normal labour. First trimester samples
generally consist of villous and extravillous cytotrophoblast,
which remain as individual cells during culture, whereas
term tissues can be differentially processed to produce syn-
cytial structures or villous cytotrophoblast. The latter may
fuse during in vitro culture to form syncytial homologues.
They produce increased quantities of hCG and hPL (Kliman
et al., 1986) in culture without additional stimuli, which par-
allels the in vivo situation—syncytia express higher levels of
these peptide hormones than the underlying cytotrophoblast
(Morrish and Marusyk, 1997).
A variety of methods have been used to deal with
the problem of contamination of cell preparations by
non-trophoblast components from the villous core. The
most robust of these combine immunomagnetic bead selec-
tion with density gradient separation. The first stage may
be positive selection (e.g. CD9+ for extravillous cells) or
negative (e.g. pan-HLA class-I removal of all cells that are
not villous cytotrophoblast). The second step is particularly
useful for removing cell debris that may accompany neg-
ative selection procedures, but may be used to clean up all
cell preparations. Most preparations are >95% CK7 posi-
tive, and can be used for a variety of in vitro investigations.
A large number of regulatory interactions between the
factors produced within the placenta have been identi-
fied. These are summarised in Fig. 2, which shows that
most data is available for relatively few of the endocrine
factors—primarily GnRH and CRH (hypothalamic-type),
hCG, hPL, ␤-endorphin, oxytocin and vGH (pituitary-type),
and progesterone, activins and inhibins (gonadal-type).
There is obviously extensive cross-talk between the factors
produced within the placenta, but our knowledge is incom-
plete, and the physiology may be more complex than this
diagram indicates. Typical of this may be the control of hCG
production by cytokines such as interleukin-1␤ (IL-1␤) and
interleukin-6 (IL-6) (Yagel et al., 1989; Nishino et al., 1990).
It has become clear that some of the regulatory pathways do
 M.H.F. Sullivan / Molecular and Cellular Endocrinology 228 (2004) 103–119 107

Fig. 2. Summary of the relationships between endocrine factors and regulators in the human placenta. Known stimulatory (→) and inhibitory ( ) effects
are shown. Main sources are: Petraglia et al. (1996), Margioris et al. (1988), Cronier et al. (1999). Groupings are ‘hypothalamic’ ( ), ‘pituitary’ ( ),
‘gonadal’ ( ) and ‘other’ ( ). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

not reflect those in the HPA and HPG axes. In particular, the
control of CRH production from the placenta is stimulated
by glucocorticoids (King et al., 2001), which may depend
on novel interactions between cyclic AMP-dependent and
glucocorticoid-regulated pathways, and the tissue-specific
expression of transcription factors (Cheng et al., 2000; King
et al., 2002). In addition, some data are contradictory, in that
both progesterone (Maruo et al., 1986) and antiprogestin
(Das and Catt, 1987) may inhibit hCG production.
The placenta also contains steroidogenic enzymes
(Fig. 3), but the placenta cannot synthesise all the primary
steroid hormones of pregnancy. The primary biosynthetic
pathway, including cholesterol recruitment from LDL, and
cytochrome P450 side-chain cleavage enzyme (P450scc,
CYP11A1) is present (Albrecht and Pepe, 1990; Chaudhary
et al., 1992; Tuckey et al., 1994), leading to the produc-
tion of pregnenolone. 3␤-Hydroxysteroid dehydrogenase
(3␤-HSD) converts this to progesterone, one of the pri-
mary steroids of pregnancy. The placenta lacks the enzyme
17␣-hydroxylase/17, 20 lyase (CYP17), and thus cannot
further metabolise the progesterone to androgens or es-
trogens. The production of these steroids relies on fetal
and maternal production of dehydroepiandrosterone sul-
phate (DHEAS) and fetal 16␣-OH-DHEAS (Fig. 3). These
steroids pass to the placenta, where they are converted to
17␤-estradiol and estriol, respectively. This complex in-
teraction is shown in Fig. 3, and serves to emphasise the
relationship between maternal, fetal and placental tissues.
Placental pregnenolone could also be metabolised through
this route, although the major sources of DHEAS are the
adrenals (Fig. 3). The placenta also interconverts estrogens
through the action of 17␤-hydroxysteroid dehydrogenase
(17␤-HSD, CYP19). In vitro studies of aromatase activity
in cultured trophoblast normally involve the addition of
appropriate substrates, and the determination of estrogenic
product levels (Wunsch et al., 1986).
The placenta does not express CYP21A2 (21-hydroxy-
lase), CYP11B1 (11␤-hydroxylase) and CYP11B2 (al-
dosterone synthase), and cannot therefore produce any
corticosteroids or aldosterone (Fig. 3). The increased levels
of these steroids found in pregnancy may be derived either
de novo from maternal biosynthesis, or from the further ma-
ternal metabolism of placental progesterone. The placenta
expresses enzymes that can metabolise glucocorticoids,
most notably type-2 11␤-hydroxysteroid dehydrogenase
(11␤-HSD) (Stewart et al., 1995); this enzyme may be stim-
ulated by cyclic AMP (Sun et al., 1998), and down-regulated
by catecholamines (Sarkar et al., 2001) and steroids (Sun
et al., 1998). This converts cortisol to the relatively inactive
cortisone, and is thought to protect the fetus, and its devel-
oping HPA axis from the increased levels of glucocorticoids
present in maternal blood during pregnancy. There is there-
fore a placental–maternal system which parallels adrenal
steroid production, as well as a placental–fetal system which
parallels gonadal steroidogenesis (Fig. 3). In contrast, the
type-1 11␤-HSD enzyme is differentially regulated (Sun
et al., 1997); this applies particularly to an up-regulation by
glucocorticoids in vitro (Sun et al., 2002), which implicates
this enzyme in labour rather than pregnancy maintenance.
These interactions do not provide any information on the
intracellular mechanisms through which these effects may
be mediated, which can also be addressed in primary cell
or cell line cultures. The main regulatory information from
primary placental cells in vitro is summarised in Table 2.
The most general finding is that membrane-permeant cyclic
AMP analogues (dibutyryl (db)- or 8-bromo-derivatives)
increase the output of hCG and of progesterone from these
cultured cell preparations. This has been reported in many
108 M.H.F. Sullivan / Molecular and Cellular Endocrinology 228 (2004) 103–119

Fig. 3. The primary steroidogenic pathways of human pregnancy are outlined. The main tissues involved are the placenta ( ), fetal adrenals ( ), fetal liver
( ) and maternal adrenals ( ). Mechanisms and enzymes involved are shown in red. HSD: hydroxysteroid dehydrogenase; S: DHEA sulphotransferase;
16␣OH: 16␣-hydroxylase; S-Sul: steroid sulphatase; CYP11A1: P450 side-chain cleavage enzyme; CYP11B1: P450 11␤-hydroxylase; CYP11B2: P450
aldosterone synthase; CYP17: 17␣-hydroxylase/17, 20 lyase; CYP19: P450 aromatase; CYP21A2: P450 21-hydroxylase (Salido et al., 1990). (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

studies (e.g. Feinman et al., 1986; Zhou et al., 1987; Nulsen mal trophoblast function. Such stimulation of progesterone
et al., 1989; Ringler et al., 1989; Dodeur et al., 1990; production clearly parallels the role of cyclic AMP in other
Strauss et al., 1992; Seki et al., 1997; Matsumoto et al., steroidogenic tissues (ovary, adrenal, testis), and suggests
1998), and may clearly be taken as representative of nor- that similar mechanisms may be involved. This may be an

Table 2
Summary of trophoblast regulation
Regulator Target Nature of effect

Cyclic AMP Progesterone Positive (Feinman et al., 1986)

3␤-HSD Positive (Mason et al., 1993)
P450scc (CYP11A1) Positive (Hum and Miller, 1993)
HCG Positive (Feinman et al., 1986)
TGF-␤1 Positive (Ritvos and Eramaa, 1991)
Calcium Progesterone Negative (Zosmer et al., 1997)
HPL Positive (Zeitler et al., 1983)
Inhibin, hCG Positive (Keelan et al., 1994)
PLA2 and melittin hPL Positive (Zeitler et al., 1983, 1991)
Arachidonic acid hPL Positive (Handwerger et al., 1981)
hPL Positive (Zeitler and Handwerger, 1985)
Dexamethasone Inhibin, hCG Positive (Keelan et al., 1994)
CRH No effect (Siler-Khodr et al., 1997)
Norepinephrine hCG, progesterone Positive (Shi and Zhuang, 1993a,b)
Phorbol ester Activin, hCG Positive (Keelan et al., 1994)
HPL Positive (Harman et al., 1986)
 M.H.F. Sullivan / Molecular and Cellular Endocrinology 228 (2004) 103–119
over-simplification, as one study indicates that the effects of
dibutyryl cyclic AMP on progesterone production are more
variable (Zosmer et al., 1997). The greatest effects of db
cyclic AMP were observed when it was added to the cells
immediately the cultures were set up; a delay of 24 h sub-
stantially decreased the impact of db cyclic AMP on proges-
terone output, and this decrease in steroidogenic capacity has
been identified previously (Ferguson et al., 1986). Further-
more, the sustained addition of relatively low concentrations
of db cyclic AMP maintained progesterone production for
up to 96 h in vitro; only at higher concentrations of stimulus
could any evidence of desensitisation be obtained (Zosmer
et al., 1997). In other steroidogenic cells, desensitisation may
be found within a few hours (Sen et al., 1979; Rani et al.,
1983; Lefevre et al., 1985). This major difference can easily
be related to the different functions of the product steroids.
In the testis and ovary, steroids are produced to (a) develop
and maintain sexual differentiation and (b) to allow the
development of gametes. In both cases these are under the
primary pulsatile control of pituitary products (luteinising
hormone and follicle-stimulating hormone) as well as more
local regulators, which leads to a pulsatile hormone release.
Adrenal glucocorticoid production must also be dynamically
responsive to the body’s requirements. In contrast, proges-
terone production in pregnancy must be maintained for 9
months—it is well established that oophorectomy in the
first 2 months of pregnancy (Diczfalusy and Borell, 1961),
or administration of progesterone receptor antagonists (e.g.
RU486) (Baulieu, 1985; DeHart and Morehead, 2001) lead
to loss of the conceptus. It should thus be no surprise that
placental progesterone production is not readily desensitised
in the presence of stimulus, but rather is sustained.
The nature of the physiological stimulus for placental
progesterone production is not entirely clear. Some in vitro
findings implicate hCG (Shi et al., 1991), but local factors
including GnRH (Shi et al., 1991) and IL-1␤ (Seki et al.,
1997) may also be involved. Clearly at least one physiologi-
cal regulator of placental progesterone production increases
intracellular cyclic AMP levels, and is itself a placental prod-
uct; this implicates hCG, but more work is needed to fully
identify how placental progesterone production is controlled.
The mechanisms have been explored in a series of studies,
which have shown that the uptake and utilisation of LDL
(Chaudhary et al., 1992), cholesterol transport (Strauss et al.,
2000) and expression of CYP11A1 (P450scc) (Chaudhary
et al., 1992; Tuckey et al., 1994; Hum et al., 1995;
Yamamoto et al., 1994, 1995; Babischkin et al., 1997;
Beaudoin et al., 1997a) are upreglated during trophoblast
differentiation and by cyclic AMP analogues. The IGFs
can also increase CYP11A1 activity (Nestler, 1987, 1990),
while inhibiting aromatase (CYP19) activity (Nestler, 1990,
1993a). IL-1␤ stimulates CYP19 activity under similar con-
ditions (Nestler, 1993b). 17␤-HSD type-1 (which mainly
converts estrone to the more active 17␤-estradiol) is ex-
pressed primarily in syncytium (Beaudoin et al., 1995;
Bonenfant et al., 2000), and is up-regulated during in vitro
109
syncytialisation, whereas the type-2 enzyme is mainly cy-
totrophoblastic and decreases in vitro; these findings suggest
that these isoforms are differentially regulated (Beaudoin
et al., 1995).
5. Cell lines from human placenta
There are three main types of cell lines derived from
the human placenta; those which have arisen spontaneously
from cultured cytotrophoblast in vitro, those which have
been immortalised by in vitro transfection with viral genes,
and those from spontaneous choriocarcinomas, which have
been maintained in vitro.
While many of these cell lines have been used to inves-
tigate the control of endocrine functions, there has recently
been some debate about the precise nature of these cells. The
primary problem seems to be that of defining trophoblast
cell-types—for example, many of the choriocarcinoma cell
lines have been defined as such on the basis of their tissue
origin, and by their production of hCG, hPL or steroids. It
is now clear that other, non-trophoblast, tumour cells may
produce hCG (Duffy, 2001), so that these definitions may
not be entirely secure. Ideally, cell lines from the human pla-
centa should be cytokeratin-7 positive (trophoblastic), and
either HLA-negative (villous) or HLAG and CD9 positive
(extra-villous). Summaries of the characteristics of the main
trophoblast cell lines has been published (King et al., 2000;
Shiverick et al., 2001), which serve mainly to emphasise the
lack of rigour that has been applied to the characterisation
of many cell lines. The data from cell lines should be con-
sidered with this proviso in mind.
5.1. Spontaneous cell lines
A series of primary cell lines have been described, and
these are listed in Table 3. Most of these are spontaneous
transformants, and can be cultured for very many passages.
They may not have been completely characterised, but they
all are of placental (or chorionic villous) origin, and may
therefore reflect villous trophoblast function. The character-
isation of these cell lines has included some assessment of
endocrine functions (peptide or steroid hormone production)
(Table 3), but few studies have used them to investigate the
control mechanisms involved. They have been used exten-
sively to investigate other aspects of trophoblast function,
including migration and invasion. HT and TL cells both ex-
pressed estrogen receptor (Ho et al., 1998), but the addition
of tamoxifen was without effect on cell numbers, which was
in contrast to an antiproliferative effect on BeWo cells. The
biosynthesis of progesterone by NPC cells was inhibited
by TGF-␤1 (Luo et al., 2002), resulting from a decrease in
cholesterol transport to mitochondria. They have not been
used for any mechanistic studies of placental endocrinology,
but rather to examine other aspects of trophoblast function,
including invasion, migration and immunology.
110 M.H.F. Sullivan / Molecular and Cellular Endocrinology 228 (2004) 103–119
Table 3
Summary of data obtained from spontaneous cell lines
TL Ho et al. (1987)
NHT Nagami et al. (1991)
ED77 Diss et al. (1992)
HT Ho et al. (1994)
NPC Rong-Hao et al. (1996)
ED27 Morgan et al. (1998)
ED31 Morgan et al. (1998)
Extravillous primary lines
HTR-8 Graham et al. (1993)
HT-116 Zdravkovic et al. (1999)
CG: chorionic gonadotrophin; PL: placental lactogen; (+) positive; (−) negative; (?) not tested.
Two lines with limited life-spans have been described
(Table 3). These cells are grown from villous explants,
and have been much more fully characterised as ex-
pressing an extravillous phenotype (Graham et al., 1993;
Aboagye-Mathiesen et al., 1996b; Zdravkovic et al., 1999).
Both can be cultured for 12–15 passages, and HTR-8 cells
in particular have been used extensively to investigate the
functions of this cell population. These have concentrated
on the expected in vivo functions of these cells in human
pregnancy, including invasion, apoptosis, proliferation or
survival, cytokine production and cell–matrix interactions
rather than any possible endocrine functions. NTR-8 cells
have also been used as the parent cells for the generation of
a transformed cell line (NTR-8/SV40neo) (Graham et al.,
1993), which will be considered in more detail in the next
section of the review.
Within the last 18 months, it has become apparent that
that one of these cell lines (ED27) is not a reliable model. It
appears that this cell line became accidentally contaminated
with the WISH cell line, which has itself been contaminated
by HeLa cells. WISH was originally amniotic epithelium
(Hayflick, 1961), but now seems to be genetically indis-
tinguishable from HeLa, and this also applies ED27 cells
(Kniss et al., 2002). There are notable phenotypic differ-
ences between the cell preparations, which have not yet been
elucidated. It is not clear when the contamination occurred,
but it must be pointed out that the ED27 cell line still ex-
presses a number of markers and biochemical features that
are considered typical of human trophoblast (Kniss et al.,
2002). These cells have been used to investigate a number
of trophoblast functions (e.g. Kniss et al., 2001), but not
specifically endocrine events, so these complications do
not impact immediately on our understanding of placental
endocrinology.
5.2. Transfected cells
A series of cell lines that have been generated by specific
transfection with viral genes are listed in Table 4. All these
cell lines are positive for at least one of hCG or hPL, which is
consistent with trophoblastic origin, but more detailed char-
acterisation to identify villous (CK7+/HLA-I−/CD9−) or
CG− PL−
CG+ PL−
CG+ PL+
CG+ PL−
CG+ PL?
CG+ PL+
CG+ PL+
Not immortal: 12–15 passages CG− PL+
Not immortal: 12–15 passages CG? PL?
extravillous phenotypes (CK7+/HLAG+/CD9+) are lack-
ing. Most of these cell lines are therefore potential mod-
els for investigating the control of endocrine functions, but
generally this has not proceeded much beyond demonstra-
tion of peptide hormone production as part of the charac-
terisation (e.g. Logan et al., 1992, using SPA-26 cells). The
methods used (viral genes and transfection techniques) are
summarised in Table 5, and show there is a broad similar-
ity between the methods used. The precise place these cells
occupy in trophoblast lineages has been the subject of some
debate (Choy et al., 2000; King et al., 2000; Manyonda et al.,
2001).
We have investigated the control of hormone production
in TCL-1 cells (Seki et al., 1997), and found that db cyclic
AMP increased progesterone production from these cells.
IL-1␤ had similar effects on progesterone production, and
increased cyclic AMP levels in parallel, implying that there
might be a functional link. HCG was produced by TCL-1
cells, but none of the factors tested had any effect on its
production. We attributed this to the insertion of an SV40
sequence into the promoter region of an hCG subunit, thus
damaging the regulatory pathways but permitting a consti-
tutive synthesis. This emphasises one of the problems with
transfected cells, that alterations to the genome may affect
endocrine functions and render them less useful models for
functional studies.
5.3. Choriocarcinoma cells
The main choriocarcinoma cell lines are listed in Table 6.
The great majority of work has been performed on the old-
est cell lines, namely BeWo, JAR (Jar or JAr) (Pattillo and
Gey, 1968; Pattillo et al., 1971) and JEG (JEG-3) (Kohler
and Bridson, 1971). These choriocarcinoma cells have been
available for over 30 years, and a correspondingly large lit-
erature is available. Their characterisation depends partly
on their known origin (in vivo choriocarinoma), and partly
on their production of placental-type hormones (hCG, pro-
gesterone). In addition, BeWo cells can express mRNA for
HLAG (Risk and Johnson, 1990) and CD9 protein (Hirano
et al., 1999b), which is indicative on an extravillous pheno-
type. These degree of characterisation may not be considered
 M.H.F. Sullivan / Molecular and Cellular Endocrinology 228 (2004) 103–119 111

Table 4
Summary characterisation of transfected cell lines
SPA-26 Chou (1978a,b) CG+ PL− HLAG? CK7?
Primary first trimester cells
HP-W1 Lei et al. (1992b) CG+ PL? HLAGm+ CK7?
Primary term placental cells
HP-A1 Lei et al. (1992b) CG+ PL? HLAGm+ CK7?
Primary term placental cells
HP-A2 Lei et al., 1992b CG+ PL? HLAGm+ CK7?
Primary term placental cells
HTR-8/SV40neo Graham et al. (1993) CG+ PL− HLAG? CK7?
HTR-8 cell line CG- PL+ HLAGic+ CK7?
TCL-1 Lewis et al. (1996) CG+ PL? HLAG− CK7+
Primary term chorion cells CG? PL? HLAG+ CK7+
IST-1 Shih et al., 1998 CG− PL+ HLAG− CK7+
Primary first trimester explants
SGHPL-4 Choy and Manyonda (1998) CG+ PL+ HLAG? CK7?
Primary first trimester cells
SGHPL-5 Choy and Manyonda (1998) CG+ PL+ HLAG? CK7+
Primary first trimester cells
RSVT-2 Khoo et al. (1998) CG− PL+ HLAG? CK7+
HTR-8 cell line CG− PL+ HLAGic+ CK7?
RSVT-2/C Khoo et al. (1998) CG− PL+ HLAG? CK7+
HTR-8 cell line CG− PL+ HLAGic+ CK7?
The source and characterisation where available of parental cell for these cell lines is given to allow comparisons. CG: chorionic gonadotrophin; PL:
placental lactogen; HLAG: human leukocyte antigen-G; CK7: cytokeratin-7; (+) positive; (−) negative; (?) not tested; (m+) mRNA positive; (ic+)
intracellular protein positive.

definitive for trophoblast cells, given the earlier considera- Five cell lines have been generated from combinations
tion of these matters, so all data obtained should be taken of JEG-3 mutants and chorion leave cells, using the meth-
in this context. There are also a small number of studies ods described by Frank et al. (2000b). These cell lines have
in which choriocarcinoma tissue has been studied, showing been deposited at the Deutsche Sammlung von Mikroorgan-
that expression of the LH/hCG receptor is increased in such ismen und Zellkulturen GmbH (DSMZ; German Collection
cells (Lei et al., 1992a). of Microorganisms and Cell Cultures), and more informa-
tion is available through http://www.dsmz.de. They are all
Table 5
cytokeratin+; AC-1M32 is GFAP+, vimentin−, whereas the
Methods to generate transfected cell lines other four cell lines display the opposite phenotype. It is not
known whether the JEG mutants and hybrids are endocrino-
Sequences from SV40
TCL-1 Recombinant retrovirus ZipSV40-6—infection logically active.
HTR-8/SV40neo pSV40neo with large T early region In some studies two or three of the established choriocar-
only—electroporation cinoma cell lines are compared to gain insight into how nor-
SGHPL-4 pSV40neo with large T antigen—poly-l-ornithine mal trophoblast cells might function, and these are outlined
SGHPL-5 pSV40neo with large T antigen—poly-l-ornithine
in Table 7. In general the cells behave very similarly (e.g.
SPA-26 tsA255 temperature sensitive mutant—infection
Bahn et al., 1981; Taylor et al., 1997) suggesting that these
Sequences from other viruses
are at least consistent models for these endpoints. In some
HP-W1 Adenovirus (ori-)SV40 wild type—infection
HP-A1 Adenovirus (ori-)SV40 A209 temperature sensitive studies, cell line specific differences were observed. This is
mutant—infection clearly shown in the work of Mandl et al., 2002 (Table 7);
HP-A2 Adenovirus (ori-)SV40 A209 temperature sensitive none of these studies showed that a factor could stimulate
mutant—infection one cell line, and inhibit the same endpoint in a different cell
IST-1 Recombinant retrovirus coding E6 and E7 proteins
line, indicating that the overall differences were moderate.
from HPV (LXNS16E6E7)—infection
RSVT-2 Plasmid coding for Rous sarcoma In other investigations, more detailed studies have been
virus-T—electroporation performed on one of these cell lines, and these are listed
RSVT-2/C Plasmid coding for Rous sarcoma below. Sometimes these studies included direct compar-
virus-T—electroporation isons with primary trophoblast; these are considered in
112 M.H.F. Sullivan / Molecular and Cellular Endocrinology 228 (2004) 103–119

Table 6
Choriocarcinomas and choriocarcinoma-derived cell lines
BeWo Pattillo and Gey (1968) Choriocarcinoma
JAR Pattillo et al. (1971) Gestational choriocarcinoma
JEG Kohler and Bridson (1971) Choriocarcinoma explants—CK7+
AC1-1 + others Funayama et al. (1997) JEG mutant
ACH1P Frank et al. (2000b) Fusion of AC1-1 (JEG mutant) with term trophoblast
AC-1M32 Frank et al. (2000b)a Fusion of AC1-1 (JEG mutant) with term chorion cells—CK7+
AC-1M46 Frank et al. (2000b)a Fusion of AC1-1 (JEG mutant) with term chorion cells
AC-1M59 Frank et al. (2000b)a Fusion of AC1-1 (JEG mutant) with term chorion cells
AC-1M81 Frank et al. (2000b)a Fusion of AC1-1 (JEG mutant) with term chorion cells
AC-1M88 Frank et al. (2000b)a Fusion of AC1-1 (JEG mutant) with term chorion cells
Only the three initial cell lines are hCG-positive. The AC1 and AC derived cell lines have not been tested.
a The method described in this paper was used to generate these clones, but the clones are not described in detail in the paper.

the next section of the review, in conjunction with the
comparisons between different papers which have investi-
gated the same endpoints in choriocarcinoma or primary
cells.
BeWo cells: the production of hCG is dependent on cyclic
AMP, protein kinase C and calcium (Kaiho et al., 1984;
Futamura et al., 1987; Nishino et al., 1991), and is also
increased by LHRH (GnRH) (Kaiho et al., 1984), EGF
(Futamura et al., 1987), keratinocyte growth factor (Matsui
et al., 2000). HCG production (used as a differentiation
marker) was also increased by culture on Matrigel (Hohn
et al., 1992, 1996), and by the addition of methotrexate
(Taylor et al., 1991); the latter increased aromatase activity.
11␤-HSD activity was decreased by catecholamines (Sarkar
et al., 2001). Proliferation of BeWo cells was decreased by
estradiol or progesterone (Kaiho et al., 1984), and increased
by EGF (Sanyal and Das, 1997). Our unpublished findings
(Seki et al., 1997) showed that db cyclic AMP increased
progesterone production time and dose-dependently.
JEG-3 cells: cAMP analogues increase P450scc (CYP-
11A1) by transcription and adrenodoxin post-transcription-
ally (Brentano and Miller, 1992; Moore et al., 1992; Guo
et al., 1994; Beaudoin et al., 1997b), 3␤-HSD (Beaudoin
et al., 1997b), and TGF-␤1 production (Ritvos and Eramaa,
1991). Calcium decreased CYP11A1 and 3␤-HSD activity
(Beaudoin et al., 1997b). 17␤-HSD-1 was increased by cal-
cium and cAMP synergistically (Beaudoin et al., 1997b).
11␤-HSD was increased by cAMP, but not by protein kinase
C (Pasquarette et al., 1996). Activin A may be an autocrine
regulator of JEG-3 cell steroidogenesis (Ni et al., 2000),
and TGF-␤1 inhibited progesterone and estradiol production
(Luo et al., 2002). TNF-␣ increased estrogen and hCG pro-

Table 7
Regulation of choriocarcinoma cells
BeWo JEG-3 JAR

cAMP analogues on hCG production + + nd Chou et al. (1978)

Progesterone produced, no estrogens + + + Bahn et al. (1981)
Retinoic acid on hCG + + + Kato and Braunstein (1991)
Retinoic acid on progsterone + + No Kato and Braunstein (1991)
T3 on vGH family members +++ ++ + Nickel and Cattini (1991)
Insulin on hCG production nd + + Ren and Braunstein (1991)
␤FGF on 17␤-HSD-1 No ++ ++ Lewintre et al. (1994)
VGH and hCS RNA Present Present Present Lytras et al. (1994)
M-CSF on proliferation and hCG No No No Saito et al. (1994)
Benzo[␣]pyrene on hCG production − No nd Zhang et al. (1995)
Nitric oxide on hCG − − nd Myat et al. (1996)
cAMP on CRH promoter activity + + nd Scatena and Adler (1996)
Hypoxia on hCG − − − Strohmer et al. (1997)
Hypoxia on VEGF ++ ++ ++ Taylor et al. (1997)
Hypoxia on hCG +/− +/− +/− Taylor et al. (1997)
Tamoxifen on growth − − − Ho et al. (1998)
Steroid uptake and steroyl-sulphatase Present Present Present Ugele and Simon (1999)
Chemokines on proliferation and hCG + + + Ishii et al. (2000)
Insulin on proliferation No + ++ Mandl et al. (2002)
IGF-I on proliferation No No – Mandl et al. (2002)
Insulin on hCG + + No Mandl et al. (2002)
IGF-I on hCG No + ++ Mandl et al. (2002)
Trialkyltin compounds (endocrine disruptors) on hCG and progesterone ++ ++ ++ Nakanishi et al. (2002)
(+) positive effect; (−) negative effect; no: no effect; nd: not done.
 M.H.F. Sullivan / Molecular and Cellular Endocrinology 228 (2004) 103–119 113

duction, and decreased progesterone production (Pedersen
et al., 1995).
Jar cells: hCG production was increased by 8-bromo-ade-
nosine or db cyclic AMP (Hussa et al., 1975, 1977; Martell
and Ruddon, 1990; Sibley et al., 1991), IL-1␤ and TNF-␣
(Yanushpolsky et al., 1993), GnRH (Ertl et al., 1993), and
PGE2 (Yagel et al., 1989). This latter paper found no ef-
fect of IL-1␤ on hCG output (Yagel et al., 1989), which
differs from the later results of Yanushpolsky et al., 1993.
The reasons for this difference are not clear. Early data sug-
gested that EGF did not affect hCG production from this
cell line (Huot et al., 1981), but recent findings show that
EGF increases hCG production, through a mechanism in-
volving cross-talk with protein kinase C (Baker et al., 1998).
Aromatase activity was also increased by db cyclic AMP
(Bellino et al., 1978). Removal of calcium, as well as the
addition of the calcium ionophore A23187, decreased hCG
production (Hussa, 1977). Jar cells interact with primary
cytotrophoblasts to increase net hCG and hPL production
(Eldar-Geva et al., 1993), but the details of this cross-talk
have not been elucidated.
6. Cell lines from other species
While there is a significant body of information on en-
docrine function in pregnancy in other species, there are few
cell lines of non-human origin. This review will therefore
focus on the endocrinology of the human placenta, and of
the cell lines derived from it, although cell lines from other
species will be considered if they provide particular insights.
Placental structures may differ substantially between differ-
ent species, so direct comparisons between apparently re-
lated cell-types may not be entirely valid.
Rcho-1 cells (Faria and Soares, 1991) were developed
as a model to study rat trophoblast function, including
placental lactogen synthesis. They have also been used to
investigate endocrine functions, including the activity of
transfected human genes. Progesterone inhibits hCG pro-
duction from transfected Rcho-1 cells (Yamamoto et al.,
2001). Human CRH promoter is active in BeWo and JEG-3
cells, but not in Rcho-1 cells; this species difference is
attributed to the presence of different transcription factors
(Scatena and Adler, 1996). BT-1 trophoblastic cells from
bovine blastocyst express interferon-␶ and bovine placental
lactogen (Shimada et al., 2001).
7. Summary of studies performed and limitations
A wide range of endpoints have been investigated using
the cell lines described in this review. Many of them are
of endocrine nature, and have investigated biosynthetic or
metabolic pathways. In general terms the data obtained from
the cell lines, and from primary cells are comparable. This
overall consistency is encouraging, suggesting that, despite
the many different cellular models used, the data do reflect
in vitro functions. This is most clearly seen in the production
of hormones, particularly hCG and progesterone, which is
up-regulated by cyclic AMP analogues, and is dependent on
the presence of calcium in the media. Protein kinase C also
seems to be a positive effector in these processes. This most
clearly in comparing Table 2 (primary cells) and Table 7
(choriocarcinoma cell lines). EGF and GnRH also seem to
be generic positive factors for hormone production, and for
proliferation in the case of EGF.
There are also some clear differences between primary
trophoblast and cell lines, as indicated by the comparative
studies shown in Table 8, but there are generally fewer dif-
ferences than similarities. It must be pointed out that entirely
negative findings (e.g. M-CSF does not affect the chorio-
carcinoma cells, Saito et al., 1994) are usually published in
context of positive data (M-CSF affects primary cells); there
may therefore be more information available than has been
published, showing more differences between the cell lines
than the generally clear findings summarised in Fig. 2.

Table 8
Comparison of primary and cell line data
Similar findings
Hussa (1977), Hussa et al. (1977): calcium and cyclic AMP required for hCG production from JAR and primary cells
Bahn et al. (1981): all three choriocarcinomas resemble primary cells in the steroidogenic enzymes present
Pedersen et al. (1995): TNF-␣ has similar effects on primary and JEG-3 cells hormone production
Ritvos and Eramaa (1991): JEG-3 and primary trophoblast TGF-␤1 increased by cAMP
Sibley et al. (1991): 8Br-adenosine increased hCG production from JAR and cytotrohoblast cells
Stephanou and Handwerger (1995): the effects of retinoic acid and thyroid hormone and similar in cytotrophoblast, and in transfected BeWo cells
Conrad et al. (1996): expression of erythorpoietin similar in primary and JAR cells
Seki et al. (1997): TCL-1 cell line and primary cells respond similarly to db cyclic AMP
Sarkar et al. (2001): primary and BeWo 11␤-HSD decreased by catecholamines
Different findings
Hoshina et al. (1985): cytotrophoblast and choriocarcinoma show different patterns of HCG and hPL expression
Maruo et al. (1986): in primary cells progesterone inhibited hCG production, but was without effect in choriocarcinoma cells
Ren and Braunstein (1991): insulin increased hCG production from JEG-3 and JAR cells, but not primary cytotrophoblast
Saito et al. (1994): M-CSF induces proliferation and differentiation of normal trophoblast, but not choriocarcinoma cells
Seki et al. (1997): different responses of primary and TCL-1 cells to IL-1␤
114 M.H.F. Sullivan / Molecular and Cellular Endocrinology 228 (2004) 103–119
The greatest limitation from an endocrine perspective is
that referred to earlier, that the cellular heterogeneity of pla-
cental trophoblast is integral to the normal physiology of
this organ, and cell lines will only reflect a part of this.
8. Other trophoblast functions
Placental trophoblast also exhibit a range of non-endocrine
functions, including cell motility, invasion (Kliman and
Feinberg, 1990), homotypic fusion (formation of syncy-
tia), complex cell–cell and cell–matrix interactions, and
apoptosis. These may be under the control of factors that
are integral parts of the endocrine axes (e.g. progesterone
inhibits matrix metalloproteinase production; Shimonovitz
et al., 1998), or regulate the endocrine axes (e.g. nitric
oxide). While these aspects lie beyond the scope of this
review, the attention of readers is directed to other reviews
and papers that consider these aspects of trophoblast func-
tion in more detail. It is possible that such processes may
be modelled accurately by cell lines, as they normally in-
volve isolated cytotrophoblast rather than combinations of
different cell-types which seem to be more important in the
endocrine functions considered in this review.
Acknowledgements
I must thank the colleagues who have helped in the warit-
ing of this review by reading the manuscript or contributing
information and insights that lie beyond the published liter-
ature. In particularly I thank Drs. Mandy Donaldson, Guy
Whitley, Ashley Moffett and Hans-Georg Frank.
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