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Cell Culture Models of Trophoblast II: Trophoblast Cell Lines— A Workshop Report
Placenta (2000), 21, Supplement A, Trophoblast Research, 14, S113–S119 doi:10.1053/plac.1999.0526, available online at http://www.idealibrary.com on
METHODOLOGY Cell Culture Models of Trophoblast II: Trophoblast Cell Lines— A Workshop Report A. Kinga, L. Thomasa and P. Bischofb a
Research Group in Human Reproductive Immunobiology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK b Hopitaux Universite´ de Geneve, Dept. de Gyne´cologie et d’Obtetrique, Laboratoire d’Hormonologie, Maternite´, Boulevard de la Cluse, CH-1211 Geneve 14, Switzerland Paper accepted 20 December 1999
Trophoblast cell lines generated from normal or choriocarcinoma cells are a powerful tool for use in the understanding of trophoblast cell biology, immunology, endocrine function and placental development. To encourage the acceptance and use of these lines in the wider scientific community the IFPA agreed in Tokyo in 1998 to convene a trophoblast cell line workshop at the EPG meeting in 1999. The objectives defined by the IFPA were as follows:
decidua. Methods used to generate the lines are also diverse. Several lines have arisen spontaneously. More frequently SV40 or human papilloma virus E6 and E7 have been used and introduced with either retroviruses, poly-l-ornithine, electroporation or adenoviruses. A more recent method has derived cell lines by fusing a derivative of JEG choriocarcinoma cells with term trophoblast cells.
(a) to identify all cell lines which have been published so far; (b) to establish criteria to evaluate their potentials and weaknesses, and to find reference laboratories for this evaluation; (c) to compare their characteristics on the basis of the criteria as defined later in this report; (d) to define the scientific questions which can be answered using these trophoblast cell lines.
CRITERIA USED TO EVALUATE THE TROPHOBLAST CELL LINES
This report summarises the discussion at the Schladming Workshop.
IDENTIFICATION OF TROPHOBLAST CELL LINES The human trophoblast cell lines which have been described in the literature are demonstrated in Table 1. These cell lines have been divided into three main groups: (a) cell lines generated from normal tissue; (b) cell lines generated from malignant tissue; (c) cell lines generated from embryonal carcinomas which have evidence of trophoblast differentiation. The source of tissue used is shown in Table 2. It is apparent that there are great differences in the tissue or cells used as a starting point. Lines have been generated from cell outgrowths from villous explants or single cell isolates from chorionic villous samples, first trimester, term placentae or chorio0143–4004/00/0A0S113+07 $35.00/0
The published criteria which have been used to define the cell lines are shown in Table 3(a). These phenotypic characteristics have been tabulated so that the most frequently used are shown on the left of the table (hCG) and the least frequent on the right. From this table, it can be seen that the most widely used markers to establish the ‘trophoblast’ nature of the cell lines are human chorionic gonadotrophin (hCG), human placental lactogen (hPL), cytokeratins, vimentin and HLA class I molecules. However, a number of problems arise when these phenotypic markers are used to define trophoblast cell lines. hCG There are several reports which show that hCG expression is a common feature of tumour cell lines (Loke, 1978; Acevedo et al., 1997). Indeed, hCG has been viewed as a model of a fetal antigen whose re-expression in tumours is a frequent occurrence (Bagshawe, 1983). In addition, cell lines are generally mononuclear whereas in vivo hCG secretion is almost entirely confined to the villous syncytiotrophoblast and is not a feature of any mononuclear trophoblast cells. hPL Although less well documented, hPL expression, like hCG, has also been reported in a range of tumours and should 2000 IFPA and Harcourt Publishers Ltd
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Table 1. ‘Trophoblast’ cell lines described in the literature Table 1(a). Human trophoblast cell lines Cell line
Name
Reference
*TCL-1 *IST-1 *HT *TL *NPC *NHT *SGHPL-4 *SGHPL-5 *SGHPL-6 *SGHPL-7 HTR-8 HTR-8/Svneo RSVT-2 RSVT2/C HT-116 ED77 ED27 ED31 SPA-26+7 lines HP-W1 HP-A1 HP-A2
Sullivan, MHF Shih, IM Ho, C-K Ho, C-K Rong Hao, L Tominaga, T Manyonda, IT Manyonda, IT Manyonda, IT Manyonda, IT Lala, PK Lala, PK Lala, PK Lala, PK Zdravkovic, M Kniss, DA Kniss, DA Kniss, DA Chou, JY Chou, JY Chou, JY Chou, JY
Lewis (1996) Shih (1998) Ho (1994) Ho (1987) Rong Hao (1996) Negami (1991) Choy (1998) Choy (1998) Unpublished Unpublished Graham (1993) Graham (1993) Khoo (1998) Khoo (1998) Zdravkovic (1999) Diss (1992) Morgan (1998) Morgan (1998) Chou (1978) Lei (1992) Lei (1992) Lei (1992)
Table 1(b). Malignant choriocarcinoma cell lines Cell line
Reference
BeWo BeWo MC-1 BeWo MC-2 JAR JEG AC1 AC1-5 AC1-9 *AC1-1 *AC-1M59 *AC-1M32 *AC-1M46 *AC-1M81 *AC-1M88
Pattillo (1968) Crescimanno (1996) Crescimanno (1996) Pattillo (1971) Kohler (1971) Funayama (1997) Funayama (1997) Funayama (1997) Funayama (1997) Frank (2000) Frank (2000) Frank (2000) Frank (2000) Frank (2000)
Table 1(c). Embryonal lines with trophoblast differentiation Cell line
Reference
H9 NCR-G3 HT-H NCCIT NCC-EC-3
Thomson (1998) Maruyama (1996) Izhar (1986) Damjanov (1993) Teshima (1988)
similarly be viewed as an onco-developmental antigen (Heyderman et al., 1985; Campo et al., 1989; Sheth et al., 1997). Unlike hCG, expression in vivo is found in extravillous trophoblast, particularly the endovascular trophoblast (Kurman et al., 1984). Cytokeratin Several reports have now confirmed that many cells at the maternal–fetal interface besides trophoblast stain with antipan-cytokeratin Mabs (Khong et al., 1986; Beham et al., 1988; Neudeck et al., 1997; Haigh et al., 1999). There was agreement during the meeting that the most useful keratin to stain trophoblast was anti-cytokeratin 7. Unlike other cytokeratins, cytokeratin 7 is not expressed by any other cells in the placental villus or maternal decidua apart from uterine glandular epithelium. Vimentin The epithelial nature of trophoblast indicates that there should be no reactivity with antibodies to other intermediate filaments, such as vimentin, desmin and to smooth muscle -actin and myosin. There are, however, reports that interstitial trophoblast may acquire vimentin reactivity (Loke and Butterworth, 1987) and, since they undergo a process resembling epithelial–mesenchymal transition, this might be expected. Nevertheless, lack of reactivity for these markers would in general be expected in trophoblast. Trophoblast cells which are vimentin positive would probably also show other features of interstitial trophoblast such as HLA-G reactivity. HLA class I molecules The two main trophoblast subpopulations, villous and extravillous, differ in their expression of HLA class I molecules (Loke and King, 1995). Villous trophoblast is devoid of any HLA class I expression at the protein level, whereas extravillous trophoblast expresses HLA-G, HLA-C and possibly HLA-E. The use of a pan anti-HLA class I Mab such as W6/32 will therefore define overall HLA class I or class I + cells. Lack of HLA class I expression could be used as evidence of a villous trophoblast origin. However, loss of HLA class I expression is a frequent occurrence in virally transformed cells (Gogusev et al., 1988), and thus lack of reactivity with W6/32 does not provide definitive evidence of villous trophoblast origin. For extravillous trophoblast evidence of positive staining for HLA class I molecules with W6/32 does not discriminate between the classical HLA class I molecules, HLA-A and HLA-B expressed by most nucleated cells, and the trophoblast HLA class I molecules HLA-C, HLA-G and HLA-E. COMPARISON OF TROPHOBLAST CELL LINES The lines marked with an asterisk in Table 1 were donated to our two laboratories for a preliminary attempt at screening
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Table 2. Source of cells and methods used to generate trophoblast cell lines Table 2(a). Human trophoblast cell lines Cell line
Source
Method of immortalization
*TCL-1
Term choriodecidua, single cell isolation
*IST-1 *HT *TL *NPC *NHT *SGHPL-4 *SGHPL-5 *SGHPL-6 *SGHPL-7 HTR-8 HTR-8/SVneo
1st trimester villous explants Term placenta, single cell isolation Term placenta, single cell isolation 1st trimester, single cell isolation 1st trimester, single cell isolation 1st trimester, single cell isolation 1st trimester, single cell isolation Not described Not described 1st trimester villous explants HTR-8
RSVT-2 RSVT2/C HT-116 ED77
HTR-8 HTR-8 1st trimester, single cell isolation Chorionic villus sample, 1st trimester, single cell isolation Chorionic villus sample, 1st trimester, single cell isolation Chorionic villus sample, 1st trimester, single cell isolation 1st trimester single cell isolation Term placenta, single cell isolation Term placenta, single cell isolation Term placenta, single cell isolation
ZipSV40-6 recombinant retrovirus, with the SV40 temp independent large T antigen LXSN16E6E7 recombinant reterovirus encoding HPV E6 and E7 proteins Spontaneous transformation Spontaneous transformation Spontaneous transformation Spontaneous transformation Poly-L-ornithine with pSV40neo plasmid containing large T-antigen of SV40 Poly-L-ornithine with pSV40neo plasmid containing large T-antigen of SV40 Not described Not described Mortal: 12–15 passages Electroporation with pSV3neo containing early region of SV40 encoding the SV40 Tag Electroporation with pRSV-T: mortal, no more than 40 passages Electroporation with pRSV-T: immortal Mortal: 12–15 passages Spontaneous transformation
ED27 ED31 SPA-26+7 lines HP-W1 HP-A1 HP-A2
Spontaneous transformation Spontaneous transformation Infection Infection Infection Infection
with with with with
temperature sensitive mutant tsA255 (mutant of SV40) adenovirus(ori-)SV40wt adenvirus(ori-)SV40tsA209 adenvirus(ori-)SV40tsA209
Table 2(b). Malignant choriocarcinoma cell lines Cell line
Source
Method of immortalization
BeWo BeWo MC-1 BeWo MC-2 JAR JEG AC1 AC1-5 AC1-9 *AC1-1 *AC-1M59 *AC-1M32 *AC-1M46 *AC-1M81 *AC-1M88
ChorioCa BeWo BeWo Gestational ChorioCa ChorioCa explants JEG (HGPRT-) JEG (HGPRT-) JEG (HGPRT-) JEG (HGPRT-) JEG/Term trophoblast JEG/Term trophoblast JEG/Term trophoblast JEG/Term trophoblast JEG/Term trophoblast
Naturally occurring Naturally occurring Naturally occurring Naturally occurring Naturally occurring Naturally occurring Naturally occurring Naturally occurring Naturally occurring Fusion between AC1-1 Fusion between AC1-1 Fusion between AC1-1 Fusion between AC1-1 Fusion between AC1-1
(JEG (JEG (JEG (JEG (JEG
mutant) mutant) mutant) mutant) mutant)
and and and and and
term term term term term
trophoblast trophoblast trophoblast trophoblast trophoblast
cells cells cells cells cells
Table 2(c). Embryonal carcinoma cell lines Cell line
Source
H9 NCR-G3 HT-H NCCIT NCC-EC-3
Inner cell mass from blastocyst Testicular embryonal carcinoma Teratocarcinoma cell line, 2061H Non-seminomatous germ cell tumour Testicular teratoma
Inner cell mass cultured on mouse fibroblast feeder layers Not described Long term culture of 2061H Explants from a medastinal tumour Explants from a testicular tumour
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Table 3. Criteria used to characterize trophoblast cell lines Table 3(a). Human trophoblast cell lines Cell line
hCG hPL CK CK-18 CK-8 CK-7 Vimentin W6/32 HLA-G
*TCL-1 *IST-1 *HT *TL *NPC *NHT *SGHPL-4 *SGHPL-5 *SGHPL-6 *SGHPL-7 HTR-8 HTR-8/Svneo RSVT-2 RSVT2/C HT116 ED77 ED27 ED31 SPA-26+7 lines HP-W1 HP-A1 HP-A2
+ + + + + + n n + n n n + + + + + + +
n + n + + n n + n + + n + + + n n n n
+ + + + + + n + n n + + + + + + + + n n n n
n + n n + n + n n + + n n n + + + n n n n
n n n n n n n n n n + + n n n + + + n n n n
n + n n n n n n n n n n n n n n n n n n n n
n + + n n n n n n n n n n n
n n + n n n + + n n + + + + + n n n n n n n
n (4H84) n n n n n n n n 87G (IC)+ n n n 87G (IC)+ n n n n mRNA mRNA mRNA
PAP LCA vWF CD56 Karyotype EMA n n + + n n n n n n n n n n n n n n n n n n
n n n n n n n n n n n n n n n n n n n n
n n n n n n n n n n n n n n n n n n n n
n n n n n n n n n n n n n n n n n n
n n Abnormal Abnormal Y Cr Abnormal n n n n n n n n n n n n Y Cr n n n
n n n n n n n n n n n n n n n n n n n n n
PAP: Placental alkaline phosphatase; LCA: leukocyte common antigen (CD45); EMA: epithelial membrane antigen; vWF: Von Willebrand Factor; Y Cr: Y chromosome; IC: intracellular; n: not tested.
Table 3 (b). Malignant choriocarcinoma cell lines
Table 3(c). Embryonal lines with trophoblast differentiation
Cell line
hCG
Cell line
hCG
CK
PAP
Vimentin
Alpha-fetoprotein
BeWo BeWo MC-1 BeWo MC-2 JAR JEG AC1 AC1-5 AC1-9 *AC1-1 *AC-1M59 *AC-1M32 *AC-1M46 *AC-1M81 *AC-1M88
+ + + n n n n n n n n n
H9 NCR-G3 HT-H NCCIT NCC-EC-3
+ + + + +
n + + + +
n n n + +
n + n + n
+ n n + n
n: not tested.
using the criteria outlined in Table 4. The NHT cell line could not be revived by either the contributing laboratory or by our laboratories. The IST-1 line has proved difficult to culture, an experience reiterated by others at the workshop. The NPC line was received too late for any detailed analysis. Of the other lines, TCL-1, HT, TL and SGHPL-4, -5, -6 and -7 were screened by flow cytometry and immunocytochemistry. Importantly, the results using this preliminary panel of
n: not tested.
markers were inconclusive and it proved impossible to be certain whether any of the cell lines tested was trophoblast or not. Ideally, it was felt that the minimum characteristic requirements for a villous trophoblast cell line would be CK7 + HLA-class I and CD9 , and for an extravillous cell line such requirements would be CK7 + , HLA-G + and CD9 + . However, none of the cell lines tested fulfilled these criteria. For example, one of the cell lines tested showed negligible HLA-class I expression but was CK7 and CD9 + . No expression of HLA-G was found in any line tested although some were W6/32 positive and indeed when tested in more detail some lines showed reactivity for HLA-A and HLA-B but no reactivity for HLA-G. The expression of these classical HLA class I antigens makes it doubtful that these particular lines are trophoblast.
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Table 4. Suggested criteria for screening trophoblast (1) Non-trophoblast cells Cell type
Marker
Reference
Leukocyte Macrophages Endothelial cells
CD45* CD14* QB-END/40* VWF Factor VIII Human milk-fat globule mucin (HMFG-2)
Kam, 1999 Goyert, 1988 Drake, 1991 Jaffe, 1977
Uterine epithelium Smooth muscle cells Fibroblasts Stromal cells All cells except trophoblast Marcophages and endothelial cells
Desmin Vimentin PDGFR* Fibroblast Surface protein* Tu¨ 155* (HLA-A, HLA-B) HLA Class II
Macdonald, 1988 Gatter, 1982 Debus, 1983 Aboagye-Mathiesen, 1996 Raines, 1990 Singer, 1989 Hutter, 1996 Glover, 1987 Mutin, 1997
(2) Trophoblast cells Marker
Trophoblast subset
Other cell types staining
References
HLA-G* GB25* F71.1
EVT Villous Villous
None None Weak on uterine epithelium
Pan cytokeratin
All
CK-7 BC-1*
All EVT
CD9* W6/32* (Pan HLA-ClassI) Tu¨ 149* (HLA-B, HLA-C)
EVT EVT EVT
Fetal mesenchyme Uterine epithelium Endothelial cells Uterine epithelium Weakly villous endothelial cells Macrophages All cells except villous trophoblast All cells except villous trophoblast All cells except villous trophoblast
Loke, 1997 Hsi, 1986 Antczak, 1987 Shorter, 1993 Aboagye-Mathiesen, 1996 Kam, 1999 Loke, 1992 Hirano, 1999 Barnstable, 1978 Hutter, 1996
*, Surface marker.
FUTURE DIRECTIONS
Table 5. Trophoblast-specific genes encoding transcription factors
There was much discussion about how the characterization of the trophoblast cell lines should proceed. The IFPA are committed to supporting further studies and it was considered probable that funding should be sought for this venture. Meanwhile it is hoped that interested laboratories could reach a consensus on the phenotypic markers and the antibodies used to define these. Other possible markers to define: (1) the trophoblast nature of a cell line and (2) the type of trophoblast cell were considered, given the inconclusive results obtained in the lines submitted using the preliminary panel proposed. However, no other markers were suggested which would satisfy the requirements of defining a trophoblast cell. Integrins, growth factor receptors and metalloproteinases were not considered useful because most are expressed on a variety of decidual and placental cell types and the expression on trophoblast will also alter during the generation of the cell line.
Human
Reference
Mouse
Reference
CDX2 HASH2 ETS1 HB24 DLX4 MOX2 MSX2
Ishii, 1998 Alders, 1997 Luton, 1997 Quinn, 1997 Quinn, 1998 Quinn, 1997 Quinn, 1997
Cdx2 Mash-2
Beck, 1995 Guillemot, 1995
Gcm1 Hand1
Basyuk, 1999 Cross, 1995 Cserjesi, 1995 Morasso, 1999 Morasso, 1999 Yamamoto, 1998 Wilkinson, 1990 Morasso, 1999
4311 Dlx3 Ets2 Pem1 Esx1
S118
It was also suggested that a small number of laboratories could isolate RNA for distribution and analysis of gene expression by RT-PCR. The possibility of screening for trophoblast specific transcription factors in this way is interesting (Table 5). Other genes which could also be analysed from RNA are CIITA and HLA class I genes (Steimle et al., 1994; Morris et al., 1998). The expression of a series of genes
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that are preferentially expressed (or absent) in the trophoblast using RT-PCR may generate preliminary data which, with the immunocytochemistry and FACS analysis, could be used for a grant application. The use of functional parameters such as invasion assays was considered premature until the problems with characterization had been more widely accepted and debated.
ACKNOWLEDGEMENTS We would like to thank M. H. F. Sullivan, I. M. Shih, C.-K. Ho, R. H. Li, T. Tominaga, H.-G. Frank, I. Manyonda and G. Whitley for contributing their lines. The authors are grateful for the stimulating comments of all the participants at the workshop.
REFERENCES Aboagye-Mathiesen G, Laugesen J, Zdravkovic M & Ebbesen P (1996) Isolation and characterization of human placental trophoblast subpopulations from first-trimester chorionic villi. Clin Diagn Lab Immunol, 3, 14–22. Acevedo HF, Hartsock RJ & Maroon JC (1997) Detection of membraneassociated human chorionic gonadotropin and its subunits on human cultured cancer cells on the nervous system. Cancer Det Preven, 21, 295–303. Alders M, Hodges M, Hadjantonakis AK, Postmus J, van Wijk I, Bliek J, de Meulemeester M, Westerveld A, Guillemot F, Oudejans C, Little P & Mannens M (1997) The human Achaete-scute homologue 2 (ASCL2, HASH2) maps to chromosome 11p15.5, close to IGF2 and is expressed in extravillous trophoblasts. Hum Mol Genet, 66, 859–867. Antczak DF, Poleman JC, Stenzler LM, Volsen SG & Allen WR (1987) Monoclonal antibodies to equine trophoblast. Troph Res, 2, 199–214. Bagshawe KD (1983) Human chorionic gonadotropin as a model for a fetal antigen. In Fetal Antigens and Cancer, Ciba Foundation Symposium 96, pp. 146–159. Barnstable CJ, Bodmer WF, Brown G, Galfre G, Milstein C, Williams AF & Zeigler A (1978) Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens—new tools for genetic analysis. Cell, 14, 9–20. Basyuk E, Cross JC, Corbin J, Nakayama H, Hunter P, NaitOumesmar B & Lazzarini RA (1999) Murine Gcm1 gene is expressed in a subset of placental trophoblast cells. Dev Dyn, 214, 303–311. Beck F, Erler T, Russell A & James R (1995) Expression of Cdx-2 in the mouse embryo and placenta: possible role in patterning of the extra-embryonic membranes. Develop Dynamikcs, 204, 219–227. Beham A, Denk H & Desoye G (1988) The distribution of intermediate filament proteins, actin and desmoplakins in human placental tissue as revealed by polyclonal and monoclonal antibodies. Placenta, 9, 479–492. Campo E, Algalsa F, Palacin A, Germa R, Sole-balcells FJ & Cardesa A (1989) Placental proteins in high-grade urothelial neoplasmus. Cancer, 63, 2497–2504. Chou JY (1978) Establishment of clonal human placental cells synthesizing human choriogonadotrophin. Proc Natl Acad Sci USA, 75, 1854–1858. Choy MY & Manyonda IT (1998) The phagocytic activity of human first trimester trophoblast. Hum Reprod, 13, 2941–2949. Crescimanno C, Foidart JM, Noel A, Polette M, Maquoi E, Birembaut P, Baramova E, Kaufmann P & Castellucci M (1996) Cloning of choriocarcinoma cells shows that invasion correlates with expression and activation of gelatinase A. Exp Cell Res, 227, 240–251. Cross JC, Flannery ML, Blanar MA, Steingrimsson E, Jenkins NA, Copeland NG, Rutter WJ & Werb Z (1995) Hxt encodes a basic helix-loop-helix transcription factor that regulates trophoblast cell development. Development, 121, 2513–2523. Cserjesi P, Brown D, Lyons GE & Olsen EN (1995) Expression of the novel basic helex-loop-helix gene eHAND in neural crest derivatives and extraembryonic membranes during mouse development. Dev Biol, 170, 664–678. Damjanov I, Horvat B & Gibas Z (1993) Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor derived cell line, NCCIT. Lab Invest, 68, 220–232. Debus E, Weber K & Osborn M (1983) Monoclonal antibodies to desmin, the muscle specific intermediate filament protein. EMBO J, 2, 2305–2312.
Diss EM, Gabbe SG, Moore JW & Kniss DA (1992) Study of thomboxane and protacyclin metabolism in an in vitro model of first trimester human trophoblast. Am J Obstet Gynecol, 167, 1046–1052. Drake BL & Loke YW (1991) Isolation of endothelial cells from human first trimester decidua using immunomagnetic beads. Hum Reprod, 6, 1156–1159. Frank HG, Gunawan B, Ebeling-Stark I, Schulten HJ, Funayama H, Cremer U, Huppertz B, Gaus G, Kaufmann P & Fuzesi L (2000) Cytogenetic and DNA-fingerprint characterization of choriocarcinoma cell lines and a trophoblast/choriocarcinoma cell hybrid. Cancer Genet Cytogenet, 116, 16–22. Funayama H, Gaus G, Ebeling I, Takayama M, Fu¨zesi L, Huppertz B, Kaufmann P & Frank HG (1997) Parent cells for trophoblast hybridization II: ACI and related trophoblast cell lines, a family of HGPRT-negative mutants of the choriocarcinoma cell line JEG-3. Troph Res, 10, 191–201. Gatter KC, Abdulaziz Z, Beverley P, Corvalan JRF, Ford C, Lane EB, Mota M, Nash JRG, Pulford K, Stein H, Taylor-Papadimitriou J, Woodhouse C & Mason DY (1982) Use of monoclonal antibodies for the histopathological diagnosis of human malignancy. J Clin Pathol, 35, 1253–1267. Glover DM, Braunstein D, Burchett S, Larsen A & Wilson CB (1987) Expression of HLA class II antigens and secretion of interleukin-1 by monocytes and macrophages from adults and neonates. Immunology, 61, 195–210. Gogusev J, Teutsch B, Morin MT, Mongiat F, Haguenau F, Suskind G & Rabotti GF (1988) Inhibition of HLA class I antigen and mRNA expression induced by Rous sarcoma virus in transformed human fibroblasts. Proc Natl Acad Sci USA, 85, 203–207. Goyert SM, Ferrero E, Rettig WJ, Yenamandra AK, Obata F & Le Beau MM (1988) The CD14 monocyte differentiation antigen maps to a region encoding growth factors and receptors. Science, 239, 497–500. Graham CH, Hawley TS, Hawley RG, MacDougall JR, Kerbel RS, Khoo N & Lala PK (1993) Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res, 206, 204–211. Guillemot F, Caspary T, Tilghman SM, Copeland NG, Gilbert DJ, Jenkins NA, Anderson DJ, Joyner AL, Rossant J & Nagy A (1995) Genomic imprinting of Mash2, a mouse gene required for trophoblast development. Nat Genet, 9, 235–242. Haigh T, Chen C-P, Jones CJP & Aplin JD (1999) Studies of mesencymal cells from 1st trimester human placenta: expression of cytokeratin outside the trophoblast lineage. Placenta, 20, 615–625. Heyderman E, Chapman DV, Richardson TC, Calvert I & Rosen SW (1985) Human chorionic gonadotrophin and human placental lactogen in extragonodal tumors. Cancer, 56, 2674–2682. Hirano T, Higuchi T, Veda M, Inoue T, Kataoka N, Maeda M, Fujiwara H & Fujii S (1999) CD9 is expressed in extravillous trophoblasts in association with integrin 3 and integrin 5. Mol Hum Reprod, 5, 162–167. Ho C-K, Li S-Y, Wang C-C, Chiang H & Wang S-Y (1994) Characterisation of a human tumorogenic, poorly differentiated trophoblast cell line. In Vitro Cell Dev Biol, 30A, 415–417. Ho C-K, Chiang H, Li S-Y, Yuan C-C & Ng H-T (1987) Establishment and characterisation of a tumorogenic trophoblast-like cell line from a human placenta. Cancer Res, 44, 3220–3224. Hsi B-L & Yeh C-JG (1986) Monoclonal antibody GB25 recognises human villous trophoblasts. Am J Reprod Immunol Microbiol, 12, 1–3.
King et al.: Trophoblast Cell Lines Hutter H, Hammer A, Blaschitz A, Hartmann M, Ebbesen P, Dohr G, Ziegler A & Uchanska-Ziegler B (1996) Expression of HLA-class I molecules in human first trimester and term placenta trophoblast. Cell Tissue Res, 286, 439–447. Ishii M, Hayakawa S, Karasaki-Suzuki M, Tohgo Y & Satoh K (1998) Expression of HOX genes in human placenta. Japan Society for Immunology of Reproduction. Proceedings of 12th Annual Meeting JSIR, pp. 103–104. December 1997. Izhar M, Siebert PD, Oshima RG, De Wolf WC & Fukuda MN (1986) Trophoblastic differentiation of human teratocarcinoma cell line HT-H. Develop Biol, 116, 510–518. Jaffe EA (1977) Endothelial cells and the biology of Factor VIII. New Eng J Med, 296, 377–383. Kam EPY, Gardner L, Loke YW & King A (1999) The role of trophoblast in the physiological change in decidual spiral arteries. Hum Reprod, 14, 2131–2138. Khong TY, Lane EB & Robertson WB (1986) An immunocytochemical study of fetal cells at the maternal-placental interface using monoclonal antibodies to keratins, vimentin and desmin. Cell Tissue Res, 246, 189–195. Khoo NKS, Bechberger JF, Shepherd T, Bond SL, McCrae KR, Hamilton GS & Lala PK (1998) SV40 Tag transformation of the normal invasive trophoblast rersults in a premalignant phenotype. I. Mechanisms responsible for hyperinvasiveness and resistance to anti-invasive action of TGF. Int J Cancer, 77, 429–439. Kohler PO & Bridson WE (1971) Isolation of hormone-producing clonal lines of human choriocarcinoma. J Clin Endocr, 32, 683–687. Kurman RJ, Main CS & Chen H-C (1984) Intermediate trophoblast: a distinctive form of trophoblast with specific morphological, biochemical and functional features. Placenta, 5, 349–370. Lei K-J, Gluzan Y, Pan C-J & Chou JY (1992) Immortalization of virus-free human placental cells that express tissue-specific functions. Mol Endocrin, 6, 703–712. Lewis MP, Clements M, Takeda S, Kirby PL, Seki H, Lonsdale LB, Sullivan MHF, Elder MG & White JO (1996) Partial characterization of an immortalized human trophoblast cell-line, TCL-1, which possesses a CSF-1 autocrine loop. Placenta, 17, 137–146. Loke YW (1978) Immunology and Immunopathology of the Human Foetal–Maternal Interaction. North Holland: Elsevier. Loke YW & Butterworth BH (1987) Heterogeneity of human trophoblast populations. In Immunoregulation and Fetal Survival (Eds) Gill TJ & Wegmann TG, pp. 197–209. New York: Oxford University Press. Loke YW & King A (1995) Trophoblast expression of major histocompatibility complex class I antigens. In Human Implantation, Cell Biology and Immunology, pp. 82–101. Cambridge: Cambridge University Press. Loke YW, Hsi B-L, Bulmer JM, Grivaux C, Hawley S, Gardner L, King A & Carter NP (1992) Evaluation of a monoclonal antibody BC-1 which identifies an antigen expressed on the surface of extravillous trophoblast. Am J Reprod Immunol, 27, 77–81. Loke YW, King A, Burrows T, Gardner L, Bowen M, Hiby S, Howlett S, Holmes N & Jacobs D (1997) Evaluation of trophoblast HLA-G antigen with a specific monoclonal antibody. Tissue Antigens, 50, 135–146. Luton D, Sibony O, Oury JF, Blot P, Dieterlen-Lievre F & Pardanaud L (1997) The c-ets1 protoonocogene is expressed in human trophoblast during the first trimester of pregnancy. Early Hum Dev, 47, 147–156. Macdonald F, Bird R, Stokes H, Russell B & Crocher J (1988) Expression of CEA, CA125, CA19-9 and human milk fat globule membrane antigen in ovarian tumours. J Clin Pathol, 41, 260–264. Maruyama T, Umezawa A, Kusakari S, Kikuchi H, Nozaki M & Hata J-I (1996) Heat shock induces differentiation of human embryonal carcinoma cells into tropectoderm lineages. Exp Cell Res, 224, 123–127. Morasso MI, Grinberg A, Robinson G, Sargent TD & Mahon K (1999) Placental failure in mice lacking the homeobox gene Dlx3. Proc Natl Acad Sci USA, 96, 162–167. Morgan M, Kniss D & McDonnel S (1998) Expression of metalloproteinases and their inhibitors in human trophoblast continuous cell lines. Exp Cell Res, 242, 18–26. Morris AC, Riley JL, Fleming WH & Boss JM (1998) MHC class II gene silencing in trophoblast cells is caused by inhibition of CIITA expression. Am J Reprod Immunol, 40, 385–394.
S119 Mutin M, Dignat-George F & Sampol J (1997) Immunologic phenotype of cultured endothelial cells: quantitative analysis of cell surface molecules. Tissue Antigens, 50, 449–458. Negami AI, Kamitani N & Tominaga T (1991) Establishment of a human trophoblastic cell line (NHT cell line) from normal early pregnancy: formation of pseudovillus structure in vitro. In Placenta: Basic Research for Clinical Application (Ed.) Soma H, pp. 68–83. Karger, Basel: Int. Conf. on Placenta, Tokyo. Neudeck H, Oei SL, Stiemer B, Hopp H & Graf R (1997) Binding of antibodies against high and low molecular weight cytokeratin proteins in the human placenta with special reference to infarcts, proliferation and differentiation processes. Histochem J, 29, 419–430. Pattillo RA, Gey GO, Delfs E & Mattingly RF (1968) Human hormone production in vitro. Science, 159, 1467–1469. Pattillo RA, Ruckert A, Hussa R, Bernstein R & Delfs E (1971) The jar cell line—continuous human multihormone production and controls. In Vitro, 6, 398–399. Quinn LM, Johnson BV, Nicholl J, Sutherland GR & Kalionis B (1997) Isolation and identification of homeobox genes from the human placenta including a novel member of the distal-less family, DLX4. Gene, 187, 55–61. Quinn LM, Latham SE & Kalionis B (1998) A distal-less class homeobox gene, DLX4, is a candidate for regulating epithelial-mesenchymal cell interactions in the human placenta. Placenta, 19, 87–93. Raines EW, Bowen-Pope DF & Ross R (1990) Platelet-derived growth factor. In Peptide Growth Factors and their Receptors I (Eds) Sporn MB & Roberts AB, pp. 173–262. Berlin: Springer-Verlag. Rong Hao L, Luo S & Zhuang LZ (1996) Establishment and characterisation of a cytotrophoblast cell line from normal placenta of human origin. Hum Reprod, 11, 1328–1333. Sheth NA, Suraiya JN, Sheth AR, Ranadive KJ & Jussawalla DJ (1977) Ectopic production of human placental lactogen by human breast tumors. Cancer, 39, 1693–1699. Shih I-M, Wang T-L, Wu T-C, Kurman RJ & Gearhart JD (1998) Expression of Mel-CAM in implantation site intermediate tropoblastic cell line, IST-1, limits its migration on uterine smooth muscle cells. J Cell Sci, 11, 2655–2664. Shorter SC, Starkey PM, Ferry BL, Clover LM, Sargent IL & Redman CWG (1993) Antigenic heterogeneity of human cytotrophoblast and evidence for the transient expression of MHC class I antigens distinct from HLA-G. Placenta, 14, 571–582. Singer KH, Scearce RM, Tuck DT, Whichard LP, Denning SM & Haynes BF (1989) Removal of fibroblasts from human epithelial cell cultures with use of a complement fixing monoclonal antibody reactive with human fibroblasts and monocytes/macrophages. J Invest Dermatol, 92, 166–170. Steimle V, Siegrist C-A, Mottet A, Losowska-Grospierre B & Mach B (1994) Regulation of MHC class II expression by interferon- mediated by the transactivator gene CIITA. Science, 265, 106–109. Teshima S, Shimosato Y, Hirohashi S, Tome Y, Hayashi I, Kanazawa H & Kakizoe T (1988) Four new human germ cell tumor cell lines. Lab Invest, 59, 328–336. Thomson JA, Itskovitz-Eidor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS & Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147. Wilkinson MF, Kleeman J, Richards J & MacLeod CL (1990) A novel oncofetal gene is expressed in a stage-specific manner in murine embryonic development. Dev Biol, 141, 451–455. Yamamoto H, Flannery ML, Kupriyanov SR, Pearce J, McKercher SR, Henkel GV, Maki RA, Web Z & Oshima RG (1998) Defective trophoblast function in mice with a targeted mutation of Ets2. Genes Dev, 12, 1315–1326. Zdravkovic M, Aboagye-Mathiesen G, Guimond M-J, Hager H, Ebbesen P & Lala PK (1999) Susceptibility of MHC class I expressing extravillous trophoblast cell lines to killing by natural killer cells. Placenta, 20, 431–440.
METHODOLOGY Cell Culture Models of Trophoblast II: Trophoblast Cell Lines— A Workshop Report A. Kinga, L. Thomasa and P. Bischofb a
Research Group in Human Reproductive Immunobiology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK b Hopitaux Universite´ de Geneve, Dept. de Gyne´cologie et d’Obtetrique, Laboratoire d’Hormonologie, Maternite´, Boulevard de la Cluse, CH-1211 Geneve 14, Switzerland Paper accepted 20 December 1999
Trophoblast cell lines generated from normal or choriocarcinoma cells are a powerful tool for use in the understanding of trophoblast cell biology, immunology, endocrine function and placental development. To encourage the acceptance and use of these lines in the wider scientific community the IFPA agreed in Tokyo in 1998 to convene a trophoblast cell line workshop at the EPG meeting in 1999. The objectives defined by the IFPA were as follows:
decidua. Methods used to generate the lines are also diverse. Several lines have arisen spontaneously. More frequently SV40 or human papilloma virus E6 and E7 have been used and introduced with either retroviruses, poly-l-ornithine, electroporation or adenoviruses. A more recent method has derived cell lines by fusing a derivative of JEG choriocarcinoma cells with term trophoblast cells.
(a) to identify all cell lines which have been published so far; (b) to establish criteria to evaluate their potentials and weaknesses, and to find reference laboratories for this evaluation; (c) to compare their characteristics on the basis of the criteria as defined later in this report; (d) to define the scientific questions which can be answered using these trophoblast cell lines.
CRITERIA USED TO EVALUATE THE TROPHOBLAST CELL LINES
This report summarises the discussion at the Schladming Workshop.
IDENTIFICATION OF TROPHOBLAST CELL LINES The human trophoblast cell lines which have been described in the literature are demonstrated in Table 1. These cell lines have been divided into three main groups: (a) cell lines generated from normal tissue; (b) cell lines generated from malignant tissue; (c) cell lines generated from embryonal carcinomas which have evidence of trophoblast differentiation. The source of tissue used is shown in Table 2. It is apparent that there are great differences in the tissue or cells used as a starting point. Lines have been generated from cell outgrowths from villous explants or single cell isolates from chorionic villous samples, first trimester, term placentae or chorio0143–4004/00/0A0S113+07 $35.00/0
The published criteria which have been used to define the cell lines are shown in Table 3(a). These phenotypic characteristics have been tabulated so that the most frequently used are shown on the left of the table (hCG) and the least frequent on the right. From this table, it can be seen that the most widely used markers to establish the ‘trophoblast’ nature of the cell lines are human chorionic gonadotrophin (hCG), human placental lactogen (hPL), cytokeratins, vimentin and HLA class I molecules. However, a number of problems arise when these phenotypic markers are used to define trophoblast cell lines. hCG There are several reports which show that hCG expression is a common feature of tumour cell lines (Loke, 1978; Acevedo et al., 1997). Indeed, hCG has been viewed as a model of a fetal antigen whose re-expression in tumours is a frequent occurrence (Bagshawe, 1983). In addition, cell lines are generally mononuclear whereas in vivo hCG secretion is almost entirely confined to the villous syncytiotrophoblast and is not a feature of any mononuclear trophoblast cells. hPL Although less well documented, hPL expression, like hCG, has also been reported in a range of tumours and should 2000 IFPA and Harcourt Publishers Ltd
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Table 1. ‘Trophoblast’ cell lines described in the literature Table 1(a). Human trophoblast cell lines Cell line
Name
Reference
*TCL-1 *IST-1 *HT *TL *NPC *NHT *SGHPL-4 *SGHPL-5 *SGHPL-6 *SGHPL-7 HTR-8 HTR-8/Svneo RSVT-2 RSVT2/C HT-116 ED77 ED27 ED31 SPA-26+7 lines HP-W1 HP-A1 HP-A2
Sullivan, MHF Shih, IM Ho, C-K Ho, C-K Rong Hao, L Tominaga, T Manyonda, IT Manyonda, IT Manyonda, IT Manyonda, IT Lala, PK Lala, PK Lala, PK Lala, PK Zdravkovic, M Kniss, DA Kniss, DA Kniss, DA Chou, JY Chou, JY Chou, JY Chou, JY
Lewis (1996) Shih (1998) Ho (1994) Ho (1987) Rong Hao (1996) Negami (1991) Choy (1998) Choy (1998) Unpublished Unpublished Graham (1993) Graham (1993) Khoo (1998) Khoo (1998) Zdravkovic (1999) Diss (1992) Morgan (1998) Morgan (1998) Chou (1978) Lei (1992) Lei (1992) Lei (1992)
Table 1(b). Malignant choriocarcinoma cell lines Cell line
Reference
BeWo BeWo MC-1 BeWo MC-2 JAR JEG AC1 AC1-5 AC1-9 *AC1-1 *AC-1M59 *AC-1M32 *AC-1M46 *AC-1M81 *AC-1M88
Pattillo (1968) Crescimanno (1996) Crescimanno (1996) Pattillo (1971) Kohler (1971) Funayama (1997) Funayama (1997) Funayama (1997) Funayama (1997) Frank (2000) Frank (2000) Frank (2000) Frank (2000) Frank (2000)
Table 1(c). Embryonal lines with trophoblast differentiation Cell line
Reference
H9 NCR-G3 HT-H NCCIT NCC-EC-3
Thomson (1998) Maruyama (1996) Izhar (1986) Damjanov (1993) Teshima (1988)
similarly be viewed as an onco-developmental antigen (Heyderman et al., 1985; Campo et al., 1989; Sheth et al., 1997). Unlike hCG, expression in vivo is found in extravillous trophoblast, particularly the endovascular trophoblast (Kurman et al., 1984). Cytokeratin Several reports have now confirmed that many cells at the maternal–fetal interface besides trophoblast stain with antipan-cytokeratin Mabs (Khong et al., 1986; Beham et al., 1988; Neudeck et al., 1997; Haigh et al., 1999). There was agreement during the meeting that the most useful keratin to stain trophoblast was anti-cytokeratin 7. Unlike other cytokeratins, cytokeratin 7 is not expressed by any other cells in the placental villus or maternal decidua apart from uterine glandular epithelium. Vimentin The epithelial nature of trophoblast indicates that there should be no reactivity with antibodies to other intermediate filaments, such as vimentin, desmin and to smooth muscle -actin and myosin. There are, however, reports that interstitial trophoblast may acquire vimentin reactivity (Loke and Butterworth, 1987) and, since they undergo a process resembling epithelial–mesenchymal transition, this might be expected. Nevertheless, lack of reactivity for these markers would in general be expected in trophoblast. Trophoblast cells which are vimentin positive would probably also show other features of interstitial trophoblast such as HLA-G reactivity. HLA class I molecules The two main trophoblast subpopulations, villous and extravillous, differ in their expression of HLA class I molecules (Loke and King, 1995). Villous trophoblast is devoid of any HLA class I expression at the protein level, whereas extravillous trophoblast expresses HLA-G, HLA-C and possibly HLA-E. The use of a pan anti-HLA class I Mab such as W6/32 will therefore define overall HLA class I or class I + cells. Lack of HLA class I expression could be used as evidence of a villous trophoblast origin. However, loss of HLA class I expression is a frequent occurrence in virally transformed cells (Gogusev et al., 1988), and thus lack of reactivity with W6/32 does not provide definitive evidence of villous trophoblast origin. For extravillous trophoblast evidence of positive staining for HLA class I molecules with W6/32 does not discriminate between the classical HLA class I molecules, HLA-A and HLA-B expressed by most nucleated cells, and the trophoblast HLA class I molecules HLA-C, HLA-G and HLA-E. COMPARISON OF TROPHOBLAST CELL LINES The lines marked with an asterisk in Table 1 were donated to our two laboratories for a preliminary attempt at screening
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Table 2. Source of cells and methods used to generate trophoblast cell lines Table 2(a). Human trophoblast cell lines Cell line
Source
Method of immortalization
*TCL-1
Term choriodecidua, single cell isolation
*IST-1 *HT *TL *NPC *NHT *SGHPL-4 *SGHPL-5 *SGHPL-6 *SGHPL-7 HTR-8 HTR-8/SVneo
1st trimester villous explants Term placenta, single cell isolation Term placenta, single cell isolation 1st trimester, single cell isolation 1st trimester, single cell isolation 1st trimester, single cell isolation 1st trimester, single cell isolation Not described Not described 1st trimester villous explants HTR-8
RSVT-2 RSVT2/C HT-116 ED77
HTR-8 HTR-8 1st trimester, single cell isolation Chorionic villus sample, 1st trimester, single cell isolation Chorionic villus sample, 1st trimester, single cell isolation Chorionic villus sample, 1st trimester, single cell isolation 1st trimester single cell isolation Term placenta, single cell isolation Term placenta, single cell isolation Term placenta, single cell isolation
ZipSV40-6 recombinant retrovirus, with the SV40 temp independent large T antigen LXSN16E6E7 recombinant reterovirus encoding HPV E6 and E7 proteins Spontaneous transformation Spontaneous transformation Spontaneous transformation Spontaneous transformation Poly-L-ornithine with pSV40neo plasmid containing large T-antigen of SV40 Poly-L-ornithine with pSV40neo plasmid containing large T-antigen of SV40 Not described Not described Mortal: 12–15 passages Electroporation with pSV3neo containing early region of SV40 encoding the SV40 Tag Electroporation with pRSV-T: mortal, no more than 40 passages Electroporation with pRSV-T: immortal Mortal: 12–15 passages Spontaneous transformation
ED27 ED31 SPA-26+7 lines HP-W1 HP-A1 HP-A2
Spontaneous transformation Spontaneous transformation Infection Infection Infection Infection
with with with with
temperature sensitive mutant tsA255 (mutant of SV40) adenovirus(ori-)SV40wt adenvirus(ori-)SV40tsA209 adenvirus(ori-)SV40tsA209
Table 2(b). Malignant choriocarcinoma cell lines Cell line
Source
Method of immortalization
BeWo BeWo MC-1 BeWo MC-2 JAR JEG AC1 AC1-5 AC1-9 *AC1-1 *AC-1M59 *AC-1M32 *AC-1M46 *AC-1M81 *AC-1M88
ChorioCa BeWo BeWo Gestational ChorioCa ChorioCa explants JEG (HGPRT-) JEG (HGPRT-) JEG (HGPRT-) JEG (HGPRT-) JEG/Term trophoblast JEG/Term trophoblast JEG/Term trophoblast JEG/Term trophoblast JEG/Term trophoblast
Naturally occurring Naturally occurring Naturally occurring Naturally occurring Naturally occurring Naturally occurring Naturally occurring Naturally occurring Naturally occurring Fusion between AC1-1 Fusion between AC1-1 Fusion between AC1-1 Fusion between AC1-1 Fusion between AC1-1
(JEG (JEG (JEG (JEG (JEG
mutant) mutant) mutant) mutant) mutant)
and and and and and
term term term term term
trophoblast trophoblast trophoblast trophoblast trophoblast
cells cells cells cells cells
Table 2(c). Embryonal carcinoma cell lines Cell line
Source
H9 NCR-G3 HT-H NCCIT NCC-EC-3
Inner cell mass from blastocyst Testicular embryonal carcinoma Teratocarcinoma cell line, 2061H Non-seminomatous germ cell tumour Testicular teratoma
Inner cell mass cultured on mouse fibroblast feeder layers Not described Long term culture of 2061H Explants from a medastinal tumour Explants from a testicular tumour
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Table 3. Criteria used to characterize trophoblast cell lines Table 3(a). Human trophoblast cell lines Cell line
hCG hPL CK CK-18 CK-8 CK-7 Vimentin W6/32 HLA-G
*TCL-1 *IST-1 *HT *TL *NPC *NHT *SGHPL-4 *SGHPL-5 *SGHPL-6 *SGHPL-7 HTR-8 HTR-8/Svneo RSVT-2 RSVT2/C HT116 ED77 ED27 ED31 SPA-26+7 lines HP-W1 HP-A1 HP-A2
+ + + + + + n n + n n n + + + + + + +
n + n + + n n + n + + n + + + n n n n
+ + + + + + n + n n + + + + + + + + n n n n
n + n n + n + n n + + n n n + + + n n n n
n n n n n n n n n n + + n n n + + + n n n n
n + n n n n n n n n n n n n n n n n n n n n
n + + n n n n n n n n n n n
n n + n n n + + n n + + + + + n n n n n n n
n (4H84) n n n n n n n n 87G (IC)+ n n n 87G (IC)+ n n n n mRNA mRNA mRNA
PAP LCA vWF CD56 Karyotype EMA n n + + n n n n n n n n n n n n n n n n n n
n n n n n n n n n n n n n n n n n n n n
n n n n n n n n n n n n n n n n n n n n
n n n n n n n n n n n n n n n n n n
n n Abnormal Abnormal Y Cr Abnormal n n n n n n n n n n n n Y Cr n n n
n n n n n n n n n n n n n n n n n n n n n
PAP: Placental alkaline phosphatase; LCA: leukocyte common antigen (CD45); EMA: epithelial membrane antigen; vWF: Von Willebrand Factor; Y Cr: Y chromosome; IC: intracellular; n: not tested.
Table 3 (b). Malignant choriocarcinoma cell lines
Table 3(c). Embryonal lines with trophoblast differentiation
Cell line
hCG
Cell line
hCG
CK
PAP
Vimentin
Alpha-fetoprotein
BeWo BeWo MC-1 BeWo MC-2 JAR JEG AC1 AC1-5 AC1-9 *AC1-1 *AC-1M59 *AC-1M32 *AC-1M46 *AC-1M81 *AC-1M88
+ + + n n n n n n n n n
H9 NCR-G3 HT-H NCCIT NCC-EC-3
+ + + + +
n + + + +
n n n + +
n + n + n
+ n n + n
n: not tested.
using the criteria outlined in Table 4. The NHT cell line could not be revived by either the contributing laboratory or by our laboratories. The IST-1 line has proved difficult to culture, an experience reiterated by others at the workshop. The NPC line was received too late for any detailed analysis. Of the other lines, TCL-1, HT, TL and SGHPL-4, -5, -6 and -7 were screened by flow cytometry and immunocytochemistry. Importantly, the results using this preliminary panel of
n: not tested.
markers were inconclusive and it proved impossible to be certain whether any of the cell lines tested was trophoblast or not. Ideally, it was felt that the minimum characteristic requirements for a villous trophoblast cell line would be CK7 + HLA-class I and CD9 , and for an extravillous cell line such requirements would be CK7 + , HLA-G + and CD9 + . However, none of the cell lines tested fulfilled these criteria. For example, one of the cell lines tested showed negligible HLA-class I expression but was CK7 and CD9 + . No expression of HLA-G was found in any line tested although some were W6/32 positive and indeed when tested in more detail some lines showed reactivity for HLA-A and HLA-B but no reactivity for HLA-G. The expression of these classical HLA class I antigens makes it doubtful that these particular lines are trophoblast.
King et al.: Trophoblast Cell Lines
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Table 4. Suggested criteria for screening trophoblast (1) Non-trophoblast cells Cell type
Marker
Reference
Leukocyte Macrophages Endothelial cells
CD45* CD14* QB-END/40* VWF Factor VIII Human milk-fat globule mucin (HMFG-2)
Kam, 1999 Goyert, 1988 Drake, 1991 Jaffe, 1977
Uterine epithelium Smooth muscle cells Fibroblasts Stromal cells All cells except trophoblast Marcophages and endothelial cells
Desmin Vimentin PDGFR* Fibroblast Surface protein* Tu¨ 155* (HLA-A, HLA-B) HLA Class II
Macdonald, 1988 Gatter, 1982 Debus, 1983 Aboagye-Mathiesen, 1996 Raines, 1990 Singer, 1989 Hutter, 1996 Glover, 1987 Mutin, 1997
(2) Trophoblast cells Marker
Trophoblast subset
Other cell types staining
References
HLA-G* GB25* F71.1
EVT Villous Villous
None None Weak on uterine epithelium
Pan cytokeratin
All
CK-7 BC-1*
All EVT
CD9* W6/32* (Pan HLA-ClassI) Tu¨ 149* (HLA-B, HLA-C)
EVT EVT EVT
Fetal mesenchyme Uterine epithelium Endothelial cells Uterine epithelium Weakly villous endothelial cells Macrophages All cells except villous trophoblast All cells except villous trophoblast All cells except villous trophoblast
Loke, 1997 Hsi, 1986 Antczak, 1987 Shorter, 1993 Aboagye-Mathiesen, 1996 Kam, 1999 Loke, 1992 Hirano, 1999 Barnstable, 1978 Hutter, 1996
*, Surface marker.
FUTURE DIRECTIONS
Table 5. Trophoblast-specific genes encoding transcription factors
There was much discussion about how the characterization of the trophoblast cell lines should proceed. The IFPA are committed to supporting further studies and it was considered probable that funding should be sought for this venture. Meanwhile it is hoped that interested laboratories could reach a consensus on the phenotypic markers and the antibodies used to define these. Other possible markers to define: (1) the trophoblast nature of a cell line and (2) the type of trophoblast cell were considered, given the inconclusive results obtained in the lines submitted using the preliminary panel proposed. However, no other markers were suggested which would satisfy the requirements of defining a trophoblast cell. Integrins, growth factor receptors and metalloproteinases were not considered useful because most are expressed on a variety of decidual and placental cell types and the expression on trophoblast will also alter during the generation of the cell line.
Human
Reference
Mouse
Reference
CDX2 HASH2 ETS1 HB24 DLX4 MOX2 MSX2
Ishii, 1998 Alders, 1997 Luton, 1997 Quinn, 1997 Quinn, 1998 Quinn, 1997 Quinn, 1997
Cdx2 Mash-2
Beck, 1995 Guillemot, 1995
Gcm1 Hand1
Basyuk, 1999 Cross, 1995 Cserjesi, 1995 Morasso, 1999 Morasso, 1999 Yamamoto, 1998 Wilkinson, 1990 Morasso, 1999
4311 Dlx3 Ets2 Pem1 Esx1
S118
It was also suggested that a small number of laboratories could isolate RNA for distribution and analysis of gene expression by RT-PCR. The possibility of screening for trophoblast specific transcription factors in this way is interesting (Table 5). Other genes which could also be analysed from RNA are CIITA and HLA class I genes (Steimle et al., 1994; Morris et al., 1998). The expression of a series of genes
Placenta (2000) Vol. 21, Supplement A, Trophoblast Research, Vol. 14
that are preferentially expressed (or absent) in the trophoblast using RT-PCR may generate preliminary data which, with the immunocytochemistry and FACS analysis, could be used for a grant application. The use of functional parameters such as invasion assays was considered premature until the problems with characterization had been more widely accepted and debated.
ACKNOWLEDGEMENTS We would like to thank M. H. F. Sullivan, I. M. Shih, C.-K. Ho, R. H. Li, T. Tominaga, H.-G. Frank, I. Manyonda and G. Whitley for contributing their lines. The authors are grateful for the stimulating comments of all the participants at the workshop.
REFERENCES Aboagye-Mathiesen G, Laugesen J, Zdravkovic M & Ebbesen P (1996) Isolation and characterization of human placental trophoblast subpopulations from first-trimester chorionic villi. Clin Diagn Lab Immunol, 3, 14–22. Acevedo HF, Hartsock RJ & Maroon JC (1997) Detection of membraneassociated human chorionic gonadotropin and its subunits on human cultured cancer cells on the nervous system. Cancer Det Preven, 21, 295–303. Alders M, Hodges M, Hadjantonakis AK, Postmus J, van Wijk I, Bliek J, de Meulemeester M, Westerveld A, Guillemot F, Oudejans C, Little P & Mannens M (1997) The human Achaete-scute homologue 2 (ASCL2, HASH2) maps to chromosome 11p15.5, close to IGF2 and is expressed in extravillous trophoblasts. Hum Mol Genet, 66, 859–867. Antczak DF, Poleman JC, Stenzler LM, Volsen SG & Allen WR (1987) Monoclonal antibodies to equine trophoblast. Troph Res, 2, 199–214. Bagshawe KD (1983) Human chorionic gonadotropin as a model for a fetal antigen. In Fetal Antigens and Cancer, Ciba Foundation Symposium 96, pp. 146–159. Barnstable CJ, Bodmer WF, Brown G, Galfre G, Milstein C, Williams AF & Zeigler A (1978) Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens—new tools for genetic analysis. Cell, 14, 9–20. Basyuk E, Cross JC, Corbin J, Nakayama H, Hunter P, NaitOumesmar B & Lazzarini RA (1999) Murine Gcm1 gene is expressed in a subset of placental trophoblast cells. Dev Dyn, 214, 303–311. Beck F, Erler T, Russell A & James R (1995) Expression of Cdx-2 in the mouse embryo and placenta: possible role in patterning of the extra-embryonic membranes. Develop Dynamikcs, 204, 219–227. Beham A, Denk H & Desoye G (1988) The distribution of intermediate filament proteins, actin and desmoplakins in human placental tissue as revealed by polyclonal and monoclonal antibodies. Placenta, 9, 479–492. Campo E, Algalsa F, Palacin A, Germa R, Sole-balcells FJ & Cardesa A (1989) Placental proteins in high-grade urothelial neoplasmus. Cancer, 63, 2497–2504. Chou JY (1978) Establishment of clonal human placental cells synthesizing human choriogonadotrophin. Proc Natl Acad Sci USA, 75, 1854–1858. Choy MY & Manyonda IT (1998) The phagocytic activity of human first trimester trophoblast. Hum Reprod, 13, 2941–2949. Crescimanno C, Foidart JM, Noel A, Polette M, Maquoi E, Birembaut P, Baramova E, Kaufmann P & Castellucci M (1996) Cloning of choriocarcinoma cells shows that invasion correlates with expression and activation of gelatinase A. Exp Cell Res, 227, 240–251. Cross JC, Flannery ML, Blanar MA, Steingrimsson E, Jenkins NA, Copeland NG, Rutter WJ & Werb Z (1995) Hxt encodes a basic helix-loop-helix transcription factor that regulates trophoblast cell development. Development, 121, 2513–2523. Cserjesi P, Brown D, Lyons GE & Olsen EN (1995) Expression of the novel basic helex-loop-helix gene eHAND in neural crest derivatives and extraembryonic membranes during mouse development. Dev Biol, 170, 664–678. Damjanov I, Horvat B & Gibas Z (1993) Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor derived cell line, NCCIT. Lab Invest, 68, 220–232. Debus E, Weber K & Osborn M (1983) Monoclonal antibodies to desmin, the muscle specific intermediate filament protein. EMBO J, 2, 2305–2312.
Diss EM, Gabbe SG, Moore JW & Kniss DA (1992) Study of thomboxane and protacyclin metabolism in an in vitro model of first trimester human trophoblast. Am J Obstet Gynecol, 167, 1046–1052. Drake BL & Loke YW (1991) Isolation of endothelial cells from human first trimester decidua using immunomagnetic beads. Hum Reprod, 6, 1156–1159. Frank HG, Gunawan B, Ebeling-Stark I, Schulten HJ, Funayama H, Cremer U, Huppertz B, Gaus G, Kaufmann P & Fuzesi L (2000) Cytogenetic and DNA-fingerprint characterization of choriocarcinoma cell lines and a trophoblast/choriocarcinoma cell hybrid. Cancer Genet Cytogenet, 116, 16–22. Funayama H, Gaus G, Ebeling I, Takayama M, Fu¨zesi L, Huppertz B, Kaufmann P & Frank HG (1997) Parent cells for trophoblast hybridization II: ACI and related trophoblast cell lines, a family of HGPRT-negative mutants of the choriocarcinoma cell line JEG-3. Troph Res, 10, 191–201. Gatter KC, Abdulaziz Z, Beverley P, Corvalan JRF, Ford C, Lane EB, Mota M, Nash JRG, Pulford K, Stein H, Taylor-Papadimitriou J, Woodhouse C & Mason DY (1982) Use of monoclonal antibodies for the histopathological diagnosis of human malignancy. J Clin Pathol, 35, 1253–1267. Glover DM, Braunstein D, Burchett S, Larsen A & Wilson CB (1987) Expression of HLA class II antigens and secretion of interleukin-1 by monocytes and macrophages from adults and neonates. Immunology, 61, 195–210. Gogusev J, Teutsch B, Morin MT, Mongiat F, Haguenau F, Suskind G & Rabotti GF (1988) Inhibition of HLA class I antigen and mRNA expression induced by Rous sarcoma virus in transformed human fibroblasts. Proc Natl Acad Sci USA, 85, 203–207. Goyert SM, Ferrero E, Rettig WJ, Yenamandra AK, Obata F & Le Beau MM (1988) The CD14 monocyte differentiation antigen maps to a region encoding growth factors and receptors. Science, 239, 497–500. Graham CH, Hawley TS, Hawley RG, MacDougall JR, Kerbel RS, Khoo N & Lala PK (1993) Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res, 206, 204–211. Guillemot F, Caspary T, Tilghman SM, Copeland NG, Gilbert DJ, Jenkins NA, Anderson DJ, Joyner AL, Rossant J & Nagy A (1995) Genomic imprinting of Mash2, a mouse gene required for trophoblast development. Nat Genet, 9, 235–242. Haigh T, Chen C-P, Jones CJP & Aplin JD (1999) Studies of mesencymal cells from 1st trimester human placenta: expression of cytokeratin outside the trophoblast lineage. Placenta, 20, 615–625. Heyderman E, Chapman DV, Richardson TC, Calvert I & Rosen SW (1985) Human chorionic gonadotrophin and human placental lactogen in extragonodal tumors. Cancer, 56, 2674–2682. Hirano T, Higuchi T, Veda M, Inoue T, Kataoka N, Maeda M, Fujiwara H & Fujii S (1999) CD9 is expressed in extravillous trophoblasts in association with integrin 3 and integrin 5. Mol Hum Reprod, 5, 162–167. Ho C-K, Li S-Y, Wang C-C, Chiang H & Wang S-Y (1994) Characterisation of a human tumorogenic, poorly differentiated trophoblast cell line. In Vitro Cell Dev Biol, 30A, 415–417. Ho C-K, Chiang H, Li S-Y, Yuan C-C & Ng H-T (1987) Establishment and characterisation of a tumorogenic trophoblast-like cell line from a human placenta. Cancer Res, 44, 3220–3224. Hsi B-L & Yeh C-JG (1986) Monoclonal antibody GB25 recognises human villous trophoblasts. Am J Reprod Immunol Microbiol, 12, 1–3.
King et al.: Trophoblast Cell Lines Hutter H, Hammer A, Blaschitz A, Hartmann M, Ebbesen P, Dohr G, Ziegler A & Uchanska-Ziegler B (1996) Expression of HLA-class I molecules in human first trimester and term placenta trophoblast. Cell Tissue Res, 286, 439–447. Ishii M, Hayakawa S, Karasaki-Suzuki M, Tohgo Y & Satoh K (1998) Expression of HOX genes in human placenta. Japan Society for Immunology of Reproduction. Proceedings of 12th Annual Meeting JSIR, pp. 103–104. December 1997. Izhar M, Siebert PD, Oshima RG, De Wolf WC & Fukuda MN (1986) Trophoblastic differentiation of human teratocarcinoma cell line HT-H. Develop Biol, 116, 510–518. Jaffe EA (1977) Endothelial cells and the biology of Factor VIII. New Eng J Med, 296, 377–383. Kam EPY, Gardner L, Loke YW & King A (1999) The role of trophoblast in the physiological change in decidual spiral arteries. Hum Reprod, 14, 2131–2138. Khong TY, Lane EB & Robertson WB (1986) An immunocytochemical study of fetal cells at the maternal-placental interface using monoclonal antibodies to keratins, vimentin and desmin. Cell Tissue Res, 246, 189–195. Khoo NKS, Bechberger JF, Shepherd T, Bond SL, McCrae KR, Hamilton GS & Lala PK (1998) SV40 Tag transformation of the normal invasive trophoblast rersults in a premalignant phenotype. I. Mechanisms responsible for hyperinvasiveness and resistance to anti-invasive action of TGF. Int J Cancer, 77, 429–439. Kohler PO & Bridson WE (1971) Isolation of hormone-producing clonal lines of human choriocarcinoma. J Clin Endocr, 32, 683–687. Kurman RJ, Main CS & Chen H-C (1984) Intermediate trophoblast: a distinctive form of trophoblast with specific morphological, biochemical and functional features. Placenta, 5, 349–370. Lei K-J, Gluzan Y, Pan C-J & Chou JY (1992) Immortalization of virus-free human placental cells that express tissue-specific functions. Mol Endocrin, 6, 703–712. Lewis MP, Clements M, Takeda S, Kirby PL, Seki H, Lonsdale LB, Sullivan MHF, Elder MG & White JO (1996) Partial characterization of an immortalized human trophoblast cell-line, TCL-1, which possesses a CSF-1 autocrine loop. Placenta, 17, 137–146. Loke YW (1978) Immunology and Immunopathology of the Human Foetal–Maternal Interaction. North Holland: Elsevier. Loke YW & Butterworth BH (1987) Heterogeneity of human trophoblast populations. In Immunoregulation and Fetal Survival (Eds) Gill TJ & Wegmann TG, pp. 197–209. New York: Oxford University Press. Loke YW & King A (1995) Trophoblast expression of major histocompatibility complex class I antigens. In Human Implantation, Cell Biology and Immunology, pp. 82–101. Cambridge: Cambridge University Press. Loke YW, Hsi B-L, Bulmer JM, Grivaux C, Hawley S, Gardner L, King A & Carter NP (1992) Evaluation of a monoclonal antibody BC-1 which identifies an antigen expressed on the surface of extravillous trophoblast. Am J Reprod Immunol, 27, 77–81. Loke YW, King A, Burrows T, Gardner L, Bowen M, Hiby S, Howlett S, Holmes N & Jacobs D (1997) Evaluation of trophoblast HLA-G antigen with a specific monoclonal antibody. Tissue Antigens, 50, 135–146. Luton D, Sibony O, Oury JF, Blot P, Dieterlen-Lievre F & Pardanaud L (1997) The c-ets1 protoonocogene is expressed in human trophoblast during the first trimester of pregnancy. Early Hum Dev, 47, 147–156. Macdonald F, Bird R, Stokes H, Russell B & Crocher J (1988) Expression of CEA, CA125, CA19-9 and human milk fat globule membrane antigen in ovarian tumours. J Clin Pathol, 41, 260–264. Maruyama T, Umezawa A, Kusakari S, Kikuchi H, Nozaki M & Hata J-I (1996) Heat shock induces differentiation of human embryonal carcinoma cells into tropectoderm lineages. Exp Cell Res, 224, 123–127. Morasso MI, Grinberg A, Robinson G, Sargent TD & Mahon K (1999) Placental failure in mice lacking the homeobox gene Dlx3. Proc Natl Acad Sci USA, 96, 162–167. Morgan M, Kniss D & McDonnel S (1998) Expression of metalloproteinases and their inhibitors in human trophoblast continuous cell lines. Exp Cell Res, 242, 18–26. Morris AC, Riley JL, Fleming WH & Boss JM (1998) MHC class II gene silencing in trophoblast cells is caused by inhibition of CIITA expression. Am J Reprod Immunol, 40, 385–394.
S119 Mutin M, Dignat-George F & Sampol J (1997) Immunologic phenotype of cultured endothelial cells: quantitative analysis of cell surface molecules. Tissue Antigens, 50, 449–458. Negami AI, Kamitani N & Tominaga T (1991) Establishment of a human trophoblastic cell line (NHT cell line) from normal early pregnancy: formation of pseudovillus structure in vitro. In Placenta: Basic Research for Clinical Application (Ed.) Soma H, pp. 68–83. Karger, Basel: Int. Conf. on Placenta, Tokyo. Neudeck H, Oei SL, Stiemer B, Hopp H & Graf R (1997) Binding of antibodies against high and low molecular weight cytokeratin proteins in the human placenta with special reference to infarcts, proliferation and differentiation processes. Histochem J, 29, 419–430. Pattillo RA, Gey GO, Delfs E & Mattingly RF (1968) Human hormone production in vitro. Science, 159, 1467–1469. Pattillo RA, Ruckert A, Hussa R, Bernstein R & Delfs E (1971) The jar cell line—continuous human multihormone production and controls. In Vitro, 6, 398–399. Quinn LM, Johnson BV, Nicholl J, Sutherland GR & Kalionis B (1997) Isolation and identification of homeobox genes from the human placenta including a novel member of the distal-less family, DLX4. Gene, 187, 55–61. Quinn LM, Latham SE & Kalionis B (1998) A distal-less class homeobox gene, DLX4, is a candidate for regulating epithelial-mesenchymal cell interactions in the human placenta. Placenta, 19, 87–93. Raines EW, Bowen-Pope DF & Ross R (1990) Platelet-derived growth factor. In Peptide Growth Factors and their Receptors I (Eds) Sporn MB & Roberts AB, pp. 173–262. Berlin: Springer-Verlag. Rong Hao L, Luo S & Zhuang LZ (1996) Establishment and characterisation of a cytotrophoblast cell line from normal placenta of human origin. Hum Reprod, 11, 1328–1333. Sheth NA, Suraiya JN, Sheth AR, Ranadive KJ & Jussawalla DJ (1977) Ectopic production of human placental lactogen by human breast tumors. Cancer, 39, 1693–1699. Shih I-M, Wang T-L, Wu T-C, Kurman RJ & Gearhart JD (1998) Expression of Mel-CAM in implantation site intermediate tropoblastic cell line, IST-1, limits its migration on uterine smooth muscle cells. J Cell Sci, 11, 2655–2664. Shorter SC, Starkey PM, Ferry BL, Clover LM, Sargent IL & Redman CWG (1993) Antigenic heterogeneity of human cytotrophoblast and evidence for the transient expression of MHC class I antigens distinct from HLA-G. Placenta, 14, 571–582. Singer KH, Scearce RM, Tuck DT, Whichard LP, Denning SM & Haynes BF (1989) Removal of fibroblasts from human epithelial cell cultures with use of a complement fixing monoclonal antibody reactive with human fibroblasts and monocytes/macrophages. J Invest Dermatol, 92, 166–170. Steimle V, Siegrist C-A, Mottet A, Losowska-Grospierre B & Mach B (1994) Regulation of MHC class II expression by interferon- mediated by the transactivator gene CIITA. Science, 265, 106–109. Teshima S, Shimosato Y, Hirohashi S, Tome Y, Hayashi I, Kanazawa H & Kakizoe T (1988) Four new human germ cell tumor cell lines. Lab Invest, 59, 328–336. Thomson JA, Itskovitz-Eidor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS & Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147. Wilkinson MF, Kleeman J, Richards J & MacLeod CL (1990) A novel oncofetal gene is expressed in a stage-specific manner in murine embryonic development. Dev Biol, 141, 451–455. Yamamoto H, Flannery ML, Kupriyanov SR, Pearce J, McKercher SR, Henkel GV, Maki RA, Web Z & Oshima RG (1998) Defective trophoblast function in mice with a targeted mutation of Ets2. Genes Dev, 12, 1315–1326. Zdravkovic M, Aboagye-Mathiesen G, Guimond M-J, Hager H, Ebbesen P & Lala PK (1999) Susceptibility of MHC class I expressing extravillous trophoblast cell lines to killing by natural killer cells. Placenta, 20, 431–440.