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EARLY
PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG |
Efficient, Rapid And Reliable Establishment Of Human Trophoblast Cell Lines Using Poly-L-Ornithine
Mei Y. Choy1, Guy St. Whitley2
and Isaac T. Manyonda1,3.
Departments of 1Immunology, 2Biochemistry & 3Obstetrics
and Gynaecology St. George's Hospital Medical School Cranmer Terrace London SW17 ORE
Short title: Trophoblast cell lines established by DNA transfection.
Key words: DNA transfection, poly-L-ornithine, extravillous trophoblast, extended lifespan
Correspondence: Dr. I T Manyonda, Division of Immunology, Department of Cellular
and Molecular Sciences, St. George's Hospital Medical School, Cranmer Terrace, London
SW17 ORE, Tel: 0181 725 3663, Fax: 0181 725 0078, email: imanyond@sghms.ac.uk
Acknowledgements: This work was supported by a WellBeing Research Grant (M2/94)
Objective
Human trophoblast cells in primary culture are difficult to use for the rigorous study of
trophoblast function because of contamination with other cell types, paucity of numbers,
poor viability and inter-experiment variation engendered by the need to prepare fresh
cells for each experiment. DNA-transfection to produce immortalized cells, or cells with
extended life-span, has been the obvious approach to solve this problem. Although there
have been a few reports in the literature describing trophoblast cell lines generated in
this way, it is clear that to date the methods are difficult and few lines have been
generated. The basic problem is that transfection efficiencies of different methods are
cell type-specific. The objectives of this study were therefore to compare the
transfection efficiencies of three commonly used techniques, to use the best technique to
generate trophoblast cell lines, and to conduct preliminary characterization studies.
Methods
We have compared calcium phosphate co-precipitation, DEAE-dextran and poly-L-ornithine
(PLO) DNA transfection protocols. We then used the most efficient to transfect human
extravillous trophoblast with pSV3neo.
Results
Our modification of the PLO method has a transfection efficiency greater than 30 times
that of the next best method. Several cell lines were established which had an extended
life span and displayed an invasive phenotype, including the expression of MHC Class I
framework antigens, human placental lactogen and human chorionic gonadotrophin, and thus
have characteristics of extravillous trophoblast. In addition these cells express the
integrin subunits b1, a1, and a3, all of which are known to be expressed in human trophoblast, and
respond to IL-1a by increased secretion of GM-CSF.
Conclusion
PLO is a highly efficient, rapid, reliable, simple and low-cost technique for the
procurement of human trophoblast cell lines which retain most, if not all, the phenotype
of the parental cell. These lines are potentially powerful tools in the rigorous study of
trophoblast function.
The rigorous investigation of human trophoblast function using cells in primary culture is hampered by contamination with other cell types, paucity of numbers, poor viability and inter-experiment variation engendered by the need to prepare fresh cells for each experiment. There have been a number of approaches to improve the purity of primary culture cells (Morgan et al., 1985; Nelson et al., 1986; Douglas and King, 1989) but while the best of these methods (flow cytometry) achieved a 97% purity (Shorter et al., 1990), it is a prohibitively expensive, labour-intensive method whose cell yield is low, and cell viability compromised by the time taken with cell-sorting. In any case, impurities of even as low as 3-10% may nevertheless compromise experiments that utilize sensitive molecular techniques, and the purity decreases with time in culture as fibroblastic over-growth predominates.
While trophoblast cell lines derived from choriocarcinoma (Kohler and Bridson, 1971; Shirotake et al., 1983) have provided valuable information about the nature of this endocrine tumour, they are not suitable models for the study of the biological and functional properties of normal and pre-neoplastic trophoblast cells. The introduction of DNA mediated gene transfer techniques over 30 years ago has to some extent circumvented many of the problems of primary cultures by taking advantage of certain DNA tumour viruses and of transforming genes to ideally immortalize, although more commonly to confer extended life-span, to cells in primary culture. This approach has provided the means to generate cell lines from cell types that are difficult to obtain and maintain in primary culture e.g lung alveolar epithelial type 2 cells (Clement et al., 1991), endothelial cells (Fickling et al., 1992) and thyroid cells (Whitley et al., 1987). Many methods have been described for introducing heterologous genes into mammalian cells. These include infection with recombinant viral vectors; complexing of DNA with diethylaminoethyl-dextran [DEAE-dextran] (McCutchan and Pagano, 1986); calcium phosphate co-precipitation (Pellicer et al., 1978); direct micro-injection (Capecchi, 1980); electroporation (Neumann et al., 1982); protoplast/spheroplast fusion (Rassoulzadegan et al., 1982); lipofection (Felger et al., 1989); laser-aided transfection by micro-puncture (Tao et al., 1987); particle bombardment (Yang et al., 1990); liposome-mediated gene transfer (Kato et al., 1991); bead transfection (Matthews et al., 1993) and poly -L-ornithine (Dong et al., 1993)
The proliferation of transfection protocols is indicative of the difficulties encountered with each protocol. Many mammalian cell types are sensitive to the treatments involved, leading to the death of limited supplies of target cells. In reality stable integration of heterologous DNA into mammalian cells can be as low as 1 in 100,000 cells (Pellicer et al., 1978; Lewis et al., 1980), and therefore the chances of a successful outcome are extremely small. Not only are there differences in transfection efficiency of the various protocols, but the efficiency of any given protocol is cell type dependent and a large variability exists between different cell types (Nairn et al., 1982; Gorman et al., 1983). There is therefore a need to optimize a transfection protocol for each cell type (Kotis et al.,1995; Teifel et al., 1997). There have now been at least three reports in the literature of the successful use of DNA transfection methodology to establish trophoblast cell lines (Graham et al., 1993; Lewis et al., 1996; Choy and Manyonda, 1998). Each group has used a different transfection protocol, usually one line is described, and it is unclear as to the ease with which two of the lines were generated, and their current fate (Graham et al., 1993; Lewis et al., 1996) (i.e whether they are still viable and available). We had limited success in producing trophoblast cell lines using the calcium phosphate co-precipitation technique, generating only one line from numerous transfections (data not shown). While Graham et al (1993) generated their line using electroporation, this method was unsuccessful in our hands, as was lipofection. However, our initial success with poly-L-ornithine compelled us to study transfection efficiencies to determine the optimal approach to the generation of human trophoblast cell lines. We have evaluated three techniques (calcium phosphate co-precipitation, DEAE-dextran and poly-L-ornithine) that are most widely used, have relatively low cytotoxicity, are technically simple and potentially easily reproducible.
Materials and MethodsMaterials
Materials were purchased from the following companies:
Hams F10, Triton -X, (Sigma Chemical Co., Poole, Dorset, UK). IL-1a, GM-CSF ELISA kits (R & D Systems Europe, Abington, UK).
Penicillin / streptomycin, Fungizone, Ultroser G, fetal calf serum (Gibco Ltd, Paisley,
Scotland, UK). 24 well plates, 60mm tissue culture plates, G418, trypsin-EDTA (Life
Technologies Ltd, Paisley, UK). Lymphoprep (Nycomed, Oslo,
Norway). CMV-ß-gal plasmid (Strategene Ltd, Cambridge, UK).
ß-gal staining kit (Invitrogen, BV, Leek, The Netherlands). hCG ELISA kit (IDS,
Tyne & Wear, UK). Nutridoma-HU (Boeringer Mannhein Biochemicals, Indianapolis,
IN, USA). Micron filters (Amicon, Beverely, MA,USA). Molecular weight standards
(Pharmacia Biotech Products, St Albans, UK). Bradford protein assay (Bio-Rad
Laboratories Ltd, Hemel Hampstead, Hertfordshire, UK)
Primary trophoblast cells
Human placental tissue was obtained from first trimester pregnancy terminations.
Samples were collected in compliance with and approval from the Local Research Ethics
Committee. A modification of the method described by Loke and Burland (Loke and Burland,
1988) was used to prepare single cell suspensions of trophoblast. Briefly, chorionic villi
in Ham’s F10 were minced finely, disaggregated with 0.25% (w/v) trypsin/ 0.02% (w/v)
EDTA for 15min at 370C with stirring, and the trypsin diluted with excess
Ham’s F10. The resultant cell suspension was filtered through two layers of muslin,
and the trophoblast cells harvested from the interface of a lymphoprep gradient. This
method yields cytotrophoblasts with a purity of 80-90%, as assessed by positive staining
for cytokeratin, hPL and MHC Class I framework antigen using the antibody W6/32, and
negative staining for vimentin.
DNA transfection protocols
i. Calcium phosphate co-precipitation
The transfection cocktail was prepared immediately prior to transfection as follows: A
fine precipitate was obtained by adding solution B (5.0mg
plasmid; 170 ml of 0.1 x Tris-EDTA [(TE) pH 8.0]; 260ml of ddH20 & 62.5 ml of 2M
CaCl2) drop-wise to solution A [500 ml of 2x Hepes
buffer saline (HBS)] while gently bubbling solution A. The transfection cocktail was left
to stand at room temperature for 30min. Transfection was undertaken on day 3 of
trophoblast cultures: cells were allowed to equilibrate for 24h prior to transfection in
Ham’s F10 containing 10% (v/v) fetal calf serum (FCS) in an incubator at 37oC
in 5 % CO2. Culture medium was replaced with the transfection cocktail and the
cultures were incubated for 6 h. At the end of the incubation, the transfection solution
was aspirated and cultures washed three times in phosphate buffered saline (PBS) before
re-culturing in Ham’s F10 with 1% (w/v) Ultroser G.
ii. DEAE-dextran-mediated transfection
On day 3 of culture, the trophoblast cells were washed with 2.0ml PBS at 37 oC.
The PBS was replaced with the transfection cocktail (5 mg
plasmid, 20 ml of 10mg/ml stock DEAE-Dextran and added to 5ml
of tris -buffered saline containing 0.2% (w/v) dextrose pH 7.4). The transfection cocktail
was distributed evenly and the culture incubated at 37oC in 5% CO2
for 30min with occasional rocking. The transfection mixture was then removed and replaced
with 2.0ml Ham’s F10 containing 1% Ultroser G and returned to the incubator for a
further 2.5 h. The medium was then aspirated and replaced with 1.0ml of culture medium
containing 10% (v/v) DMSO and left for 2.5 min. at room temperature. Finally, the DMSO
mixture was aspirated, the cultures washed thrice, and 2.0ml fresh culture medium
comprising Ham’s F10 and 1% (w/v) Ultroser G added.
iii. Poly-L-ornithine (PLO)-mediated transfection
Primary trophoblast cultures were transfected with PLO on day 3 according to the
method of Dong et al. [1993]. The culture medium was replaced with 1.0ml transfection
cocktail [5.0mg of plasmid and 10mg/ml
of PLO dispensed from a 10mg/ml stock solution] and the cells incubated for 6h at 37oC
in 5% CO2 with gentle rocking every 1.5h. The transfection cocktail was then
aspirated and replaced with 2.0ml of a 30% (v/v) DMSO in Ham’s F10 with 1% (v/v) FCS
for exactly 4 min at room temperature. The DMSO was aspirated and the cultures washed
twice in medium before adding standard culture medium (Ham’s F10 with 1% (w/v)
ultroser G) and incubated at 37oC, 5% CO2 . Optimal PLO transfection
time was determined by incubating cultures for 1,2,4 & 6 hr.
Determination of transfection efficiency
The relative efficiency of introducing DNA into cells (transfection efficiency) with
calcium phosphate co-precipitation, DEAE-Dextran and PLO was determined by using a
positive control vector, a CMV b -galactosidase-expressing
plasmid [b -gal] designed for monitoring transfected cells. b -gal plasmid contains the bacterial LacZ gene which is translated
into the b -galactosidase enzyme. Transfection efficiency of
the above methods was assayed by in situ staining for the hydrolysed product
(transfected cells stain blue) of b -galactosidase activity
using a b-gal staining kit. Extravillous trophoblast cells,
prepared according to the above method, were seeded at 1.6 x 10 6 in 60mm
plates and at 1.5 x 10 5 in 24 well plates in triplicate and transfected on day
3 of culture. In the latter, the volume of the transfection cocktail was reduced to 100ml/well whilst keeping the same concentrations of solutions and
plasmid. Cultures were stained 24 h after transfection and incubated in the staining
solution for 48h before counting under the light microscope. Each experiment was repeated
three times.
Generation of extravillous trophoblast cell lines with pSV3neo
Human extravillous trophoblast cell lines were generated using PLO mediated transfection
with a pSV3neo plasmid. This plasmid expresses large T antigen and small t antigens of
SV40 which confer an extended life span whilst the neo gene confers resistance to
the antibiotic G418, which therefore selects for stable transfectants. Cells were
seeded at a density of 1-2 x 106 in 60mm tissue culture plates and transfected
with 5mg of pSV3neo according to the PLO protocol described
above. Five days after transfection, cells incorporating the plasmid were selected by
incubation in the same culture medium, containing 0.3mg/ml G418. The medium was replaced
every three days and the G418 removed after 7 days. Cultures were continually monitored
and resistant colonies were removed using 0.25% trypsin/0.02% EDTA and expanded prior to
evaluation.
Characterisation of trophoblast cell lines
Characterization studies were carried out with cell lines at passages 15-20
Immunocytochemistry
The indirect immunoperoxidase and indirect immunofluorescence (biotin/avidin ) methods
were used as previously described (Choy and Manyonda, 1998). A panel of antibodies (see Tables 2,3,4) were used to demonstrate the trophoblastic lineage of
the cell lines.
Karyotypic analysis
This was performed on early passage trophoblast cell lines using the trypsin giemsa
banding technique.
Hormonal production
Trophoblast cell lines SGHPL-2 (1.8 x 10 4 /well), SGHPL-3 ( 3.7 x 10
4 /well ), SGHPL-4 ( 3 x 10 4 /well ) & SGHPL-5 (3.7 x 10 5 /well)
were each cultured in triplicate in 500m L Ham’s F10 with
10% FCS in 24 well plates. The supernatant was collected after 24 h, pooled and assayed
for human beta chorionic gonadotrophin (b-hCG) using an hCG
ELISA kit. Cells were trypsinized and counted at the end of the incubation and the results
from three experiments were expressed as mIU hCG/ml /106 cells.
Stimulation of GM-CSF secretion by IL-1a:
Trophoblast primary culture cells and trophoblast cell lines were seeded into 24 well
plates and treated with 1.0ng/ml of recombinant IL-1a for 3
days. Culture supernatants from treated and untreated cells were removed and assayed for
human GM-CSF using a human GM-CSF ELISA kit. The protein concentration in each well was
determined by the Bradford protein assay and GM-CSF secretion expressed as pg/mg of
protein. A total of four wells were assayed from two experiments.
Results
Primary culture human extravillous trophoblast
Purity was assessed by immunostaining with anti-cytokeratin antibody. The isolation
procedure consistently yielded trophoblast preparations that were 80%-90% pure (Figure 1a). In addition, a similar percentage of cells
were also positively stained with W6/32 (Figure 1b),
thus showing their extravillous lineage (Loke and Burland, 1988).
Transfection Efficiencies
b-galactosidase positive cells were counted in each plate or
well and transfection efficiency expressed as % transfection i.e. number of blue
cells/number of cells seeded x 100 % (Table 1). PLO
produced morphological changes in cells even before 6h that appear to be associated with
cellular toxicity. In comparison, DEAE-cultures appeared more viable, and more cells
remained after transfection (Figure 2a & 2c). Nevertheless, PLO consistently gave a higher %
transfection efficiency in both 60mm and 24 well plates (Table
1). PLO was on average 5 times more efficient than DEAE-dextran under standard
conditions. However, the 6h standard PLO transfection time was not optimal for
transfection, since a reduction in transfection time resulted in an increase in
transfection efficiency by six fold (Figure 2a &
b; Figure 3), presumably due to reduced cellular
toxicity.
Calcium phosphate co-precipitation failed to transfect trophoblast with CMV-b-gal in all of the total of six experiments (0% transfection efficiency, Table 1). This was despite retaining good viability after transfection.
Characteristics of trophoblast cell lines
Immunocytochemistry
The panel of antibodies shown in Table 2 were used. All
cell lines expressed the placental hormones hPL and hCG, and all but SGHPL-3 expressed
pregnancy specific beta-1 glycoprotein (SP1). All were negative for the macrophage marker
MAC-3 and the endothelial cell associated antigen von Willibrand factor. Cell line
SGHPL-5, one of our more recent lines, has retained cytokeratin expression in contrast to
the more common switch to vimentin. This line also expresses the integrin subunits a1, a3 and b1 (Figure 4d,e & f). All the lines express the HLA class I monomorphic
antigen recognised by w6/32 (Table 3 & 4).
Karyotype
Early passage cell lines all show a normal karyotype (Table
5).
Secretion of b -hCG
b -hCG was secreted by all the cell lines (Figure 5), with different secretion rates in different
lines: 784 mIU/ml/106 cells in SGHPL-2; 327 mIU/ml/10 6 cells in
SGHPL-3; 218 mIU/ml/10 6 cells in SGHPL-4; and 45 mIU/ml/10 6 cells
in SGHPL-5.
IL-1a stimulation of GM-CSF secretion
IL-1a stimulates GM-CSF secretion in both primary cultures
and trophoblast cell lines (Figure 6). Secretion of
GM-CSF in SGHPL-2 was beyond the maximal level of detection for
the ELISA assay and we have given an arbitrary figure of >2500pg/mg to indicate its
secretion in relation to the other cell lines.
There is a requirement for ready access to unlimited supplies of trophoblast cells in any laboratory with a major interest in trophoblast research, and the establishment of cell lines using DNA transfection technology is arguably the best way to procure such supplies. Although at least three cell lines produced by DNA transfection have now been reported in the literature (Graham et al., 1993; Lewis et al., 1996; Choy and Manyonda, 1998), different techniques have been employed, and it is clear from our own experience, and that of others, that success in establishing these lines can be unpredictable, since different transfection methods appear to be cell-type specific (Neumann et al., 1982; Kotnis et al., 1995). Calcium phosphate co-precipitation, a method which has been used for over 20 years to introduce DNA into mammalian cells (Graham et al., 1973) and which has been shown to produce high transfection efficiencies in some cells (Chen et al., 1987), nonetheless registered a zero transfection efficiency for trophoblast cells. It is of course possible that successful transfection of human trophoblast with calcium phosphate may yet be achieved with modifications and adjustments to the standard protocol. The poly-L-ornithine method, when optimised for time of incubation, produced a transfection rate that was 30 times greater than DEAE-dextran. Both are polycation-mediated transfection methods based upon their ability to neutralise negative charges on nucleic acids in order to promote DNA entry into cells. Amongst other factors, the higher transfection efficiency with poly-L-ornithine may be attributed to its ability to transfect non-proliferating trophoblasts in culture (quiescent cells).
The ability to generate in-house cell lines rapidly and reproducibly allows use of early passage cells which tend to have retained most, if not all, of the parental phenotype. While we have previously attempted to generate trophoblast cell lines using a variety of transfection techniques including electroporation and lipofection (unpublished), in this study we have evaluated those techniques that are most commonly used, are simple, easy to reproduce, and of low cost. By a study of transfection efficiency, we have found that poly-L-ornithine is vastly more efficient than both calcium phosphate co-precipitation and DEAE-dextran in the establishment of human extravillous trophoblast cell lines.
Introduction of the large T antigen of SV40 into cells in primary culture induces rapid division and an extended lifespan (Ide et al., 1984; Stein, 1990; Page et al., 1991) until a period called "crisis" begins, when there is balanced cell growth and cell death followed by a decrease in the number of surviving cells (Shay et al., 1989). Occasionally, a clone of cells emerges from the population in crisis and gives rise to an immortalized cell line, although this is a rare event (Shay et al., 1993). The cell lines described in this study have undergone more than 20 passages in culture but have not as yet manifested morphological features of crisis and therefore are not truly immortalized. The stimulation of cell growth by transfection with the early region of SV40 must be viewed with caution, as rapidly growing cells may lose some of the phenotypic characteristics of the primary cell type. For example, some of our lines switched from expressing cytokeratin to vimentin. Although these intermediate-size filaments are thought to be markers of cellular differentiation (cytokeratins for epithelial; vimentin for mesenchymal and desmin for smooth muscle cell differentiation), in fact expression of these filaments is more closely related to the growth rate of cells. Thus reversible loss of cytokeratin and increase in vimentin has been reported during rapid growth in culture of untransformed human mesothelial cells (Connel and Rheinwald, 1983). Rapidly dividing human mammary epithelial cells transformed with SV40 T-antigen have also been shown to lose cytokeratin expression (Van Der Haegen and Shay, 1993), while co-expression of cytokeratin and vimentin is now a well recognized phenomenon in normal, reactive and neoplastic states (Franke et al., 1979; Czernobilsky et al., 1985; McGuire et al., 1989) and especially in fetal tissues (Van Muijen et al., 1987). In fact, extravillous trophoblast primary cultures are known to lose cytokeratin and to express vimentin (Aboagye-Mathiesen et al., 1996). While most of our lines switched from cytokeratin to vimentin expression, SGHPL-5 has retained cytokeratin expression from inception. The significance of these changes in terms of trophoblast function remains unclear, but the cautious interpretation of any data obtained using cell lines requires that any significant experiments with cell lines be repeated with cells in primary culture to corroborate the data.
We have generated a large number of trophoblast cell lines using poly-L-ornithine, and conducted preliminary characterization of some of these lines. Karyotypic analysis of cells after the first 3-5 passages indicate that all cell lines retained a normal diploid chromosome complement. Thereafter, loss of chromosomes occurred with increasing passage, with the exception of the line SGHPL-2, which continued to retain a normal female karyotype beyond the fifteenth passage. There was no apparent pattern to chromosomal loss within and between cell lines. This change in karyotype is a well recognized phenomenon in rapidly dividing cells, and was not unique to our cell lines. However, since most of the characterization experiments were conducted with cells at approximately passage 15-20, this loss of chromosomes does not appear to affect phenotype.
Additional significant characteristics include the expression of MHC-Class 1 framework antigen (but negative for Class II), as shown by positive staining with the antibody w6/32 (figure 4b) a feature of the extravillous invasive population which has not been documented in previously reported trophoblast cell lines (Graham et al., 1993; Lewis et al., 1996). There was no apparent change with increasing passage in the expression of MHC antigens. Other trophoblastic features such as the expression of hormonal markers (Table 3) and secretion of hCG (Figure 5) further reflect the retention of characteristics of the parental cell.
Integrins are thought to play a crucial role in trophoblastic invasion, and specific integrin profiles are thought to be associated with the invasive phenotype. Thus the integrin profile on trophoblasts in normal placentation is different from that in the failed invasion seen in pre-eclampsia (PE) (Zhou et al., 1993). In normal placentation, there is down-regulation of a6b4 (a laminin receptor which is strongly expressed in villous cytotrophoblasts) and up-regulation of a5b1 as the trophoblasts differentiate into the cell column extravillous variety. In addition, invasive trophoblasts ultimately express the a1 subunit as they penetrate into the uterine wall (Damsky et al., 1992). This is also lacking in cytotrophoblasts from PE pregnancy (Lim et al., 1997). The inability of trophoblasts from PE pregnancies to undergo normal switching of integrin expression during invasion suggests that a correct integrin profile along the invasive pathway is a crucial requirement for normal placentation (Zhou et al., 1997). Our cell line SGHPL-5 strongly expresses the a1 integrin subunit (figure 4d) in contrast to the cell line described by Chou et al [1992], thus rendering our line a powerful tool for the study of the role of integrins in trophoblastic invasion. Since cytokines are also thought to be important in the regulation of trophoblast invasion, it was encouraging to note that at least as far as GM-CSF was concerned, the secretory pattern of our lines was similar to that seen in primary culture cells (Figure 6).
In summary, we show that poly-L-ornithine is a cheap, simple, reproducible and highly efficient technique of DNA transfection of human extravillous trophoblasts to establish cell lines which retain most, if not all, of the parental cell phenotype. This approach allows for an unlimited supply of pure trophoblastic cells, thus overcoming the limitations of using primary culture cells. However, we would always urge caution in the interpretation of data obtained from experiments using cell lines, and we advocate that any such data should be corroborated by data obtained from experiments with untransfected cells.
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References
Aboagye-Mathiesen, G., Laugesen, J., Zdravkovic, M. and Ebbesen, P. (1996). Isolation and characterization of human placental trophoblast subpopulations from first-trimester chorionic villi. Clin. Diagn. Lab. Immunol., 3, 14-22
Capecchi, M.R. (1980). High efficiency transformation by direct microinjection of DNA into cultured mamamlian cells. Cell, 22, 479-88
Chen, C. and Okayama, H. (1987). High efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol., 7, 2745-52
Choy, M.Y. and Manyonda, I.T. (1998). The phagocytic activity of human first trimester extravillous trophoblast. Hum. Reprod., 13, 2941-2949
Clement, A., Steele, MP., Brody, J.S. and Riedel, N. (1991). SV40-immortalized lung alveolar epithelial cells display post-transcriptional regulation of proliferative related genes. Exp. Cell. Res., 196, 198-205
Connell, N.D. and Rheinwald, J.D. (1983). Regulation of the cytoskeleton in mesothelial cells: reversible loss of keratin and increase in vimentin during rapid growth in culture. Cell, 34, 245- 253
Czernobilsky, B., Moll, R., Levy, R. and Franke, W.W. (1985). Co-expression of cytokeratin and vimentin filaments in mesothelial, granulosa, and rete ovarii cells of the human ovary. Eur. J. Cell Biol., 37, 175-190
Damsky, C.H., Fitzgerald, M.L. and Fisher, S.J. (1992). Matrix components and adhesion receptors are intricately modulated during first trimester cytotrophoblast differentiation along the invasive pathway, in vivo. J. Clin. Invest., 89, 210-222
Dong, Y., Skoultchi, A.I. and Pollard, J.W. (1993). Efficient DNA transfection of quiescent mammalian cells using poly-L-ornthine. Nucleic Acid Res., 21, 771-772
Douglas, G.C. and King, B.F. (1989). Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres. J. Immunol. Methods., 119, 259-268
Felger, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M. and Danielsen, M. (1989). Lipofection : a highly efficient , lipid mediated DNA transfection procedure . Proc. Natl. Acad. Sci. USA., 84, 7413-7417
Fickling, S.A., Tooze, J.A., Whitley, G.StJ. (1992). Characterisation of human umbilical vein endothelial cell lines produced by transfection with the early region of SV40. Exp. Cell. Res. 201: 517-521
Franke, W.W., Schmid, E., Winter, S., Osborn, M. and Weber, K. (1979). Widespread occurrence of intermediate-sized filaments of the vimentin-type in cultured cells. Exp. Cell. Res., 123, 25-46
Gorman, C., Padmanabhan, R. and Howard, B.H. (1983). High efficiency DNA-mediated transformation of primate cells. Science 221, 551-3.
Graham, C.H., Hawley, T.S., Hawley, R.G., MacDougall, J.R., Kerbel, R.S., Khoo, N. and Lala, P.K. (1993). Establishment and characterization of First Trimester Human Trophoblast Cells with Extended Lifespan. Exp. Cell. Res., 206, 204-211
Graham, F.L, and van der Eb, A.J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA to a culture of primary rodent embryo fibroblasts. Virology, 52, 456-67
Ide, T., Tsuji, Y., Nakashima, T. and Ishibashi S. (1984). Progress of aging in human dilpoid cells transformed with tsA mutant of simian virus 40. Exp. Cell. Res., 150, 321-328
Kato, K., Nakanishi, M., Kaneda, Y., Uchida, T. and Okada, Y. (1991). Expression of hepatitis B virus surface antigen in adult rat liver. Co-introduction of DNA and muclear protein by a simplified liposome method. J. Biol. Chem., 266, 3361-64
Kohler, P.O. and Bridson, W.E. (1971). Isolation of hormone-producing clonal lines of human choriocarcinoma. J. Clin. Endocrinol. Metab., 32, 683-687
Kotnis, R.A., Thompson, M.M., Eady, S.L., Budd, J.S., Bell, P.R.F. and James R.F.L. (1995). Optimisation of gene transfer into vascular endothelial cells using electroporation. Eur. J. Endovasc. Surg., 9, 71-79
Lewis, W.H., Scrinivasan, P.R., Stoke, N. and Siminovitch, L. (1980). Parameters governing the transfer of the genes for thymidine kinase and dihydrofolate reductase into mouse cells using metaphase chromosomes or DNA. Somat. Cell Mol. Genet., 6, 333-347
Lewis, M.P., Clements, M., Takeda, S., Kirby, P.L., Seki, H., Lonsdale, L.B., Sullivan, M.H.F., Elder, M.G. and White, J.O. (1996). Partial characterization of an immortalized human trophoblast cell line, TCL-1, which possesses a CSF-1 autocrine loop. Placenta, 17, 137-146
Logan, S.K., Fisher, S.J. and Damsky, C.H. (1992). Human placental cells transformed with temperature-sensitive simian virus 40 are immortalized and mimic the phentype of invasive cytotrophoblasts at both permissive and nonpermissive temperatures. Cancer Res., 52, 6001-6009
Lim, K.H., Zhou, Y., Janatpour, M., McMaster, M., Bass, K., Chun, S.H. and Fisher, S.J.( 1997). Human cytotrophoblast differentiation /invasion is abnormal in pre-elampsia. Am. J. Pathol., 151, 1809-1818
Loke, Y.W. and Burland, K. (1988). Human trophoblast cells cultured in modified medium and supported by extracellular matrix. Placenta, 9, 173-182
McCutchan, J.H. and Pagano, J.S. (1986). Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethyl amino dextran. J. Natl. Cancer Inst., 41, 351-357
McGuire, L.J., McGuire, J.P.W., Ng, J. and Lee, C.K. (1989). Coexpression of cytokeratin and vimentin. Appl.Pathol., 7, 73-84
Matthews, K.E., Mills, G.B., Horsfall, W., Hack, N., Skorecki, K. and Keating, A. (1993). Bead transfection: rapid and efficient gene transfer into marrow stromal and other adherent mammalian cells. Exp. Haematol., 21, 697-702
Morgan, D.M.L., Toothill, V.J. and Landon, M.J. (1985). Long term culture of human trophoblast cells. Br. J. Obstet. Gynaecol., 92, 84-92
Nairn, R.S., Adair, G.M. and Humphrey, R.M. (1982). DNA mediated gene transfer in Chinese hamster ovary cells; clonal variation in transfer efficiency. Mol. Gen. Genetics, 187, 384-90
Nelson, D.M., Meister, R.K., Ortman-Nabi, J., Sparks, S. and Stevens, V.C. (1986). Differentiation and secretory activities of cultured human placental trophoblast. Placenta, 7, 1- 16
Neumann, E., Schaefer-Ridder, M., Wang, Y. and Hofschneider, P.H. (1982). Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J., 1, 841-5
Page, R.A., Taylor, A.H., Whitley, G.St., Johnstone, A.P. and Nussey, S.S. (1991). Effects of aging on the growth and differentiated function of transfected human thyrocytes. Mol. Cell. Endocrinol., 82, 143-150
Pellicer, A., Wigler, M., Axel, R. and Silverstein, S. (1978). The transfer and stable integration of the HSV thymidine kinase gene into mouse cells. Cell, 14, 131-141
Rassoulzadegan, M., Binetruy, B. and Cuzin, F. (1982). High frequency of gene transfer after fusion between bacteria and eukaryotic cells. Nature, 295, 257
Shay, J.W. and Wright, W.E. (1989). Quantitation of the frequency of immortalization of normal diploid fibroblasts by SV40 large T-antigen. Exp. Cell Res., 184, 109-118
Shay, J.W., Van Der Haegen, B.A., Ying, Y. and Wright, W.E. (1993). The frequency of immortalisation of human fibroblasts and mammary epithelial cells transfected with SV40 large T antigen. Exp. Cell Res., 209, 45-52
Shirotake, S., Kaiho, T., Iwasawa, H., Kawata, M., Higaki, K., Ishige, H., Takaminizawa, H. and Minanihisamatu, M. (1983). A newly established human gestational choriocarcinoma cell line and its characterization. Gynecol. Oncol., 15, 413-21
Shorter, S.C., Jackson, M.C., Sargent, I.L., Redman, C.W.G. and Starkey, P.M. (1990). Purification of human cytototrophoblast from term amniochorion by flow cytometry. Placenta, 11, 505-513
Stein, G.H. (1985). SV40-transformed human fibroblasts: evidence for cellular aging in pre-crisis cells. J. Cell Physiol., 125, 36-44
Tao, W., Wilkinson, J., Stanbridge, E. and Berns, M.W. (1987). Direct gene transfer into human cultured cells facilitated by laser micropuncture of the cell membrane. Proc. Natl. Acad Sci. USA., 84, 4180-4
Teifel, M., Heine, Lars-Thorsten., Milbredt, S. and Freidel, P. (1997). Optimisation of transfection of human endothelial cells. Endothelium, 5, 21-35.
Van Der Haegen, G.N.P. and Shay, J.W. (1993). Immortalization of human mammary epithelial cells by SV40 large T –antigen involves a two step mechanism. In Vitro Cell Dev. Biol. Anim., 29, 180-182
Van Muijen, G.N.P., Ruiter, D.J. and Warnar, S.O. (1987). Coexpression of intermediate filament polypeptides in human fetal and adult tissues. Lab Invest., 57, 359-369
Whitley, G.St.J., Nussey, S.S. and Johnstone, A.P. (1987). SGHTL-34, a thyrotrophin-responsive immortalised human thyroid cell line generated by transfection. Mol. Cell. Endocrinol., 52, 279-284
Yang, N.S, Burkholder, J., Roberts, B., Martinell, B. and McCabe, D. (1990). In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment . Proc. Natl. Acad Sci. USA., 87, 9568-72.
Zhou, Y., Damsky, C.H., Chiu, K., Roberts, J.M. and Fisher, S. (1993). Preeclampsia is associated with abnormal expression of adhesion molecules by invasive cytotrophoblasts. J. Clin. Invest., 91, 950-960
Zhou, Y., Fisher, S.J, Janatpour, M., Genbacev, O., Dejana, E. and Wheelock, M. (1997). Human cytotrophoblasts adopt a vascular phenotype as they differentiate. J. Clin. Invest., 99, 2139-2151
Immunocytochemical staining of trophoblast primary culture
Trophoblast primary cultures consistently gave between 80%-90% pure trophoblast as demonstrated by indirect immunoperoxidase staining with anti-cytokeratin antibody (1a, x100 magnification, scale bar=20µm); the same preparation also produced a similar % of cells staining with w6/32 antibody (1b, x100 magnification, scale bar =25µm) showing these cells to be of the extravillous lineage.
In situ staining for ß-galactosidase activity
a: PLO mediated transfection (6h incubation time). Cells appear morphologically stressed and viability compromised. Transfected cells are seen as blue cells (arrows, x 40 magnification, scale bar=60µm)
b: PLO mediated transfection under 1h. The number of positive cells have greatly increased & staining of ß-galactosidase is more intense (arrows, x 40 magnification, scale bar =60µm)
c: DEAE-dextran mediated transfection; very few transfected cells (arrows) despite retaining good viability after transfection. (x 40 magnification, scale bar=60µm)
Effect of PLO transfection time on % transfection efficiency
% transfection efficiency was compared at different PLO transfection times (1h, 2h, 4h & 6h). In all three experiments, the standard 6h incubation time was the least efficient; transfection efficiency increased with decreasing transfection time such that maximum transfection was observed at 1h.
Immunofluorescene staining of cell line SCHPL-5
Indirect immunofluoresecent staining of cell line SGHPL-5 showing positive
staining with
a) anti-cytokeratin, green filamentous staining (arrow, x40 magnification, scale
bar=10µm)
b) w6/32, granular staining in cytoplasm around nucleus (arrow, x100 magnification, scale
bar=5µm) c) anti-hPL, staining areas in the cytoplasm (arrow, x100 magnification, scale
bar=5µm d) anti-a1 integrin
subunit, localised areas of very intense membrane staining (arrow, x100 magnification,
scale bar =4µm) e) anti-a3 integrin
subunit, localised to certain regions (arrow, x100 magnification, scale bar
=5µm) f) anti-ß1 integrin subunit, x100 magnification, scale bar=8µm.
Negatives controls using mouse and rabbit serum at 1/50 and 1/100 respectively showed no
specific staining (data not shown)
Secretion of ßhCG
Secretion of ß hCG by trophoblast cell lines was measured using a solid phase enzyme-linked immunosorbent assay (ELISA) kit. (minimum detectable concentration is approx. 1mIU/ml). Results presented are the mean +SD of three experiments, each experiment being assayed three times.
Effect of IL-1a on GM-CSF secretion in trophoblast primary cultures and trophoblast cell lines
The effect of IL-1alpha on basal GM-CSF secretion was measured with a human GM-CSF ELISA kit in both trophoblast primary cultures and trophoblast cell lines. Stimulation of GM-CSF secretion by I1-1alpha in cell lines SGHPL-2 went beyond the maximum limit of detection and hence we have assigned an arbitrary value of 2500 pg/mg of protein. The results presented are the mean +SD from two experiments, each carried out in duplicates.
Determination of % transfection efficiencies of PLO, DEAE-Dextran and calcium phosphate co-precipitation by in situ staining of ß-galactosidase. Each result represents the average of three experiments, counts from triplicate wells were averaged for each experiment. The pattern of % transfection efficiency between the three methods was the same from experiments in 60mm plates or 24 well plates. PLO % transfection efficiency in 60mm and 24 well plates was 4 & 6 times that of DEAE-dextran respectively
.
Shows the range of antibodies used in the immunocytochemical characterisation of trophoblast cell lines.
Indirect immunoperoxidase staining profile of cell lines SGHPL-2, SCHPL-3, SCHPL-4
Indirect immunofluorescence staining profile of cell line SGHPL-5
Karyotypic analysis of early passage cell lines.
