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Hum. Reprod. Advance Access originally published online on June 19, 2007
Human Reproduction 2007 22(8):2111-2119; doi:10.1093/humrep/dem144
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© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The isolation and characterization of a population of extravillous trophoblast progenitors from first trimester human placenta

Joanna L. James1, Peter R. Stone and Lawrence W. Chamley

Department of Obstetrics and Gynecology, University of Auckland, Private Bag 92019, Auckland 1001, New Zealand

1 Correspondence address. Tel: +649 3737599; Fax: +649 3035969; E-mail: j.james{at}auckland.ac.nz


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: It is widely accepted that most if not all villous cytotrophoblasts from term placentae are committed to differentiate into syncytiotrophoblast, but that early in gestation villous cytotrophoblasts are bipotential and capable of differentiating into either extravillous trophoblasts (EVTs) or syncytiotrophoblast. In contrast, our previous work has suggested that two separate populations of cytotrophoblast exist in the first trimester, one committed to EVT differentiation and the other to syncytiotrophoblast differentiation. In this work, we have isolated and characterized the population of ‘EVT progenitors’.

METHODS: First trimester villous explants were cultured for 10 days then subjected to sequential trypsinization. Viable cells that adhered to Matrigel following trypsinization were cultured for up to 5 days and characterized by immunohistochemistry.

RESULTS: The isolation protocol yielded >90% cytokeratin positive trophoblasts, which expressed markers characteristic of EVT progenitors. Over 5 days of culture, these isolated putative EVT progenitors did not syncytialize, but ~20% differentiated into HLA-G positive EVTs.

CONCULSIONS: It is likely that the isolated putative EVT progenitors are the population of EVT progenitors previously identified in vivo. The characteristics of these isolated putative EVT progenitors provides further evidence for separate progenitors of EVT and syncytiotrophoblast in the first trimester.

Key words: trophoblast/placenta/differentiation/cytotrophoblast/extravillous trophoblast


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The placenta is a fetal organ, i.e. responsible for nutrient and gas exchange between the mother and baby throughout pregnancy. Placental villi are encapsulated by a continuous layer of multinucleated syncytiotrophoblast, beneath which is a layer of mononuclear cytotrophoblasts. Cytotrophoblasts in the monolayer underlying the syncytiotrophoblast are able to fuse into the overlying syncytiotrophoblast to support the expansion of this layer (Boyd and Hamilton, 1970; Loke and King, 1996). Anchoring villi give rise to extravillous trophoblasts (EVTs) that grow away from the villi in columns and invade into the maternal decidua. These EVTs act to physically connect the placenta to the decidua, and to transform the maternal spiral arteries into large bore conduits capable of providing an adequate blood supply to the placenta and fetus as pregnancy progresses (Brosens, 1988Go).

It is widely accepted that most if not all villous cytotrophoblasts from term placentae are committed to differentiate into syncytiotrophoblast (Kliman et al., 1986Go; Morrish et al., 1997Go), but that early in gestation villous cytotrophoblasts are bipotential and capable of differentiating into either EVTs or syncytiotrophoblast (Loke and King, 1996; Baczyk et al., 2005Go). Despite this belief, there is little direct evidence for a bipotential villous cytotrophoblast population in first trimester placentae. Our previous work has suggested that rather than one bipotential population of cytotrophoblasts, two separate populations of cytotrophoblast exist in the first trimester, one committed to EVT differentiation and the other to syncytiotrophoblast differentiation (James et al., 2005Go). We have shown that despite the death of the majority of monolayer villous cytotrophoblasts within one week of culture, cytotrophoblasts in anchoring villi were able to survive and produce fresh EVT outgrowth for up to three weeks in culture, but did not regenerate the syncytiotrophoblast (James et al., 2005Go). We termed these cells ‘EVT progenitors’ (James et al., 2005Go). In this paper, we have studied this EVT progenitor population in greater detail by isolating them based on their ability to survive for extended periods in explant culture. These putative EVT progenitors have been cultured in vitro and their characteristics and capacity to differentiate has been investigated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
The isolation of trophoblasts from villous tissue after extended explant culture
Villus tips were gently teased from first trimester placentae from 6 to 10 weeks of gestation. Matrigel (Becton Dickinson, Sydney, Australia) was thawed slowly at 4°C and diluted to 1 10 in Dulbecco's modified Eagle's medium salts/F12 at 4°C (DMEM/F12) (Life Technologies, Auckland, New Zealand). Flasks (t25; Falcon, Sydney) were coated with 2.5 ml of 10% Matrigel and incubated at 37°C for 25 min. Excess Matrigel was removed from the flask, leaving a thin Matrigel coat over the bottom of the flask. Up to 200 villous explants (~8 mg wet weight) were randomly selected from each placenta (as previously described in James et al., 2005Go). 15 ml of complete trophoblast medium [DMEM/F12 containing 10% fetal bovine serum (FBS), 5 ng/ml epidermal growth factor, 5 µg/ml insulin, 10 µg/ml transferrin, 100 µg/ml streptomycin, 20 nM sodium selenite, 400 U/l human chorionic gonadotrophin and 100 U/l penicillin] was added and then explants in the medium were transferred to the Matrigel coated flask and cultured for 10 days at 37°C in a humidified ambient oxygen atmosphere with 5% CO2. The culture medium was replaced once during this period on day 3 or 4 of culture. Medium removed at this time was passed through a 0.22 µm filter and stored at –20°C to be used in conditioned complete trophoblast medium (75% complete trophoblast medium and 25% medium pooled from cultures of villous explants incubated in a flask of complete trophoblast medium for 3–4 days).

On day 10 of culture, flasks were agitated to dislodge explants that may have adhered to the Matrigel, and the explants were transferred to a 30 ml universal tube (Falcon, Australia) and centrifuged for 8 min at 480 x g. The supernatant was removed and 20 ml of warm 0.25% trypsin (Invitrogen, Auckland) in phosphate-buffered saline (PBS) and 0.2 ml of DNAse (4000 U/ml) (Roche, Germany) was added. Explants were incubated with the trypsin and DNAse at 37°C for 10 min and the tube was gently agitated every 2 min during this time. At the end of the 10 min period, explants were allowed to settle to the bottom of the tube and the supernatant was aspirated. Fresh trypsin and DNAse was added and this process was repeated a further four times. The first digest supernatant was discarded, but the remaining four digest supernatants were collected and filtered through cheese cloth. One millilitre of FBS and 1000 U of trypsin inhibitor were then added to each supernatant and supernatants were stored at 37°C until all the subsequent supernatants had been collected. Supernatants were then centrifuged for 8 min at 480 x g. Cell pellets from the four digest supernatants were pooled and resuspended at ~10 000 cells/ml in conditioned complete trophoblast medium. Between 50 000 and 80 000 viable cells were usually obtained from each placenta by this method as determined by trypan blue staining and counting on a haemocytometer. The isolated cells were cultured on 96-well plates coated with 10% Matrigel. After 24 h, residual cell debris was rinsed off with PBS and the tightly adherent cells were cultured in fresh conditioned complete trophoblast medium.

Examination of cell viability in villous explants by confocal microscopy
In order to determine cell viability, explants were labelled simultaneously with the fluorescent markers 5-chloromethylfluorescin diacetate (CMFDA), which is actively taken up and metabolized into a fluorescent green compound in the cytoplasm of viable cells, and ethidium bromide (EtBr) which is able to enter cells in which membrane permeability has been compromised and intercollate into the DNA, thereby staining the cell nucleus fluorescent red. A total of 12 explants from three placentae of 7–9 weeks of gestation that had been in explant culture for 10 days were incubated with 5 µM CMFDA in complete trophoblast medium at 37°C for 1 h and 30 min. Excess CMFDA was removed by washing the explants three times with complete trophoblast medium. Explants were then incubated with 2.5 µg/ml EtBr in PBS at room temperature for 1 min, and washed four times with PBS. At each timepoint, one additional explant was incubated in 5% (w/v) Virkon (Biolab, Auckland) for 10 min before staining to serve as a positive control of cell death. Jeg-3 choriocarcinoma cells in exponential growth were used as a control to indicate cell viability. Explants were visualized at room temperature by confocal microscopy.

Immunohistochemistry
Immunocytochemistry was performed in situ on cells or explants with EVT outgrowths in the wells of 96-well plates. Medium was removed from each well and cells were fixed in 100 µl of methanol for 10 min. Wells were washed once with 100 µl of PBS–Tween then non-specific binding was blocked by the addition of 100 µl of 10% normal goat serum in PBS–Tween (blocking solution) for 10 min at room temperature. Wells were then washed three times with PBS–Tween. The relevant primary antibody diluted in blocking solution (Table 1) was added to the wells for 1 h at room temperature. Control wells were incubated without addition of primary antibody. Wells were then washed three times with PBS–Tween and endogenous-peroxidase activity was quenched by the addition of 50 µl of 10% H2O2 in methanol for 5 min. Wells were then washed three times with PBS–Tween. A Zymed Histostain-Plus Kit (Invitrogen) containing biotinylated secondary antibody and enzyme conjugated to streptavidin was used according to the manufacturers' instructions and added to the wells for 10 min. Wells were washed three times with PBS–Tween and staining was developed with AEC+ or 3,3-diaminobenzidene (DAB) solution for 10–20 min. Wells were then washed with de-ionized water. Unless the relevant primary antibody was reactive with a nuclear antigen, 100 µl of haematoxylin nuclear stain was added to each well for 4 min. Wells were washed with tap water. Staining was observed immediately by phase contrast microscopy and digitally photographed. Control slides of cryosectioned first trimester placental tissue were stained alongside immunohistochemistry experiments.


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  		Table 1: Primary antibodies, dilutions and sources

 
Quantification of isolated trophoblast purity
Cultures of cells obtained by trypsin digestion, which had undergone immunocytochemistry with antibodies reactive with cytokeratin or vimentin were examined by phase contrast microscopy, and the number of cytokeratin positive and cytokeratin negative (haematoxylin stained only) cells as well as the number of vimentin positive and vimentin negative (haematoxylin stained only) cells from each placenta were counted in five fields of view using a x10 objective (x100 magnification).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
The isolation of putative EVT progenitors from villous explants
A total of 12 explants from four placentae were removed from culture prior to trypsin digestion, stained with CMFDA and EtBr, and examined by confocal microscopy to confirm that the cytotrophoblasts located in the multilayered cell islands in anchoring villous tips were the only viable cells in these explants (Fig. 1). In order to isolate the putative EVT progenitors, explanted villi that had been cultured in a flask for 10 days were subjected to sequential trypsin digestion. The cells obtained from the second to fifth sequential trypsin digests were pooled and cultured on a thin layer of Matrigel. After 24 h, there was a population of viable cells adherent to the Matrigel which will henceforth be referred to as putative EVT progenitors (Fig. 2). The vast majority of these putative EVT progenitors showed a distinctive morphology, with a rounded rectangular shape, peri-nuclear granules and large nuclei (Fig. 2). Over 4 days in culture the number of these cells increased, and small colonies of cells became evident (Fig. 2).


Figure 1
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  		Figure 1: Optical section obtained by confocal microscopy of a villus from an 8.1 week explant stained with 5 µM CMFDA and 2.5 µM EtBr and visualized by confocal microscopy after 10 days in culture. CMFDA positive cells are present in the villous tip only (arrow)

 

Figure 2
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  		Figure 2: Phase contrast photomicrographs showing putative EVT progenitors isolated from villous explants. The images show the putative EVT progenitors from day 1 to 4 (A–D, respectively) after trypsin digestion. The arrows in C provide some examples of colonies of trophoblasts. All wells were washed with PBS on day 1 (prior to photomicrography) to remove cellular debris

 
The purity of putative EVT progenitor preparations
In order to determine the purity of isolated putative EVT progenitors, the cells isolated from three placentae were assessed by immunohistochemistry using antibodies reactive with cytokeratin-7 (to label the trophoblasts) and vimentin (to label any contaminating cells) (Fig. 3). The percentage of cytokeratin or vimentin positive cells in five random fields at x10 magnification was determined over each of the first 4 days of culture. An average, the population of cells obtained were >90% trophoblasts on day 1 of culture (Fig. 3). However, as the cultures progressed rapid proliferation of the small number of contaminating cells resulted in a decline in trophoblast purity.


Figure 3
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  		Figure 3: Photomicrographs of immunohistochemistry showing that the putative EVT progenitors were trophoblasts. Putative EVT progenitors stained with cytokeratin (A). Some vimentin positive cells were also evident (B, arrow). Sections of first trimester villous tissue were stained with cytokeratin (C) or vimentin (D) as true controls

 
Isolated putative EVT progenitors express {alpha}vbeta6 integrin but not tenascin in situ
In order to determine if the isolated putative EVT progenitors expressed {alpha}vbeta6 integrin or tenascin, cultures of isolated putative EVT progenitors were stained in situ with antibodies reactive with {alpha}vbeta6 integrin or tenascin. The cells did not express tenascin, but the vast majority of them did express {alpha}vbeta6 integrin (Fig. 4). In order to confirm that {alpha}vbeta6 integrin was not expressed by EVTs, and that the putative EVT progenitors were not differentiated EVTs, EVT outgrowths from villous explants were also stained with anti-{alpha}vbeta6 integrin and tenascin in situ. No expression of {alpha}vbeta6 integrin or tenascin was seen in EVTs. Sections of fresh frozen first trimester placenta used as positive controls showed that neither {alpha}vbeta6 integrin or tenascin were expressed in floating villi, but expression of both of these markers was seen in the tips of some anchoring villi immediately adjacent to cell islands of multilayered cytotrophoblasts.


Figure 4
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  		Figure 4: Photomicrographs showing putative EVT progenitors stained after 4 days of culture with antibodies reactive with {alpha}vbeta6 integrin (A), tenascin (B) or CD9 (C) Fresh frozen first trimester placental sections were used as a positive control for {alpha}vbeta6 integrin (D) tenascin (E) and CD9 expression (F). EVT outgrowths obtained during culture of villous explants were also probed with antibodies reactive with {alpha}vbeta6 integrin (G) and tenascin (H). Negative controls were putative EVT progenitors stained with a class matched antibody (CD45) (I). Cells were counterstained with haematoxylin

 
The expression of CD9 by putative EVT progenitors
Previous reports have suggested that fibroblasts can be immunodepleted from isolated trophoblasts using CD9 as a target (Morrish et al., 1997Go; Verrijit et al., 1997Go). However, some populations of isolated trophoblasts have been reported to express CD9 (Nagamatsu et al., 2004aGo). Immunohistochemical analysis demonstrated that a significant proportion of putative EVT progenitors from three placentae were positive for CD9 after 3 days in culture (Fig. 4). Fresh frozen sections of first trimester villous tissue were used to examine the expression of CD9 in vivo. These sections showed that mesenchymal cells in the core of large villi expressed CD9, but CD9 was not expressed by all mesenchymal cells in smaller villi. Cytotrophoblasts in the monolayer underlying the syncytiotrophoblast did not express CD9, but groups of CD9 positive cells that may have been EVT progenitors were observed in some villous tips (Fig. 4C). Additional wells were stained with antibodies reactive with cytokeratin or vimentin to ensure that cultures contained >90% trophoblasts.

Putative EVT progenitors proliferate in culture
A key characteristic of cytotrophoblasts in the multilayered cell islands of anchoring villous tips is their ability to proliferate, as shown by their expression of Ki67 (Vivovac et al., 1995Go; Nishimura et al., 2004Go). Isolated putative EVT progenitors were often present on the Matrigel surface in colonies from day 2–3 of culture. Therefore, in order to determine whether these colonies of cells were forming by proliferation of the putative EVT progenitors, these cells were examined for the expression of Ki67. The majority of cells in these groups, which had the morphology of trophoblasts, stained with Ki67 (Fig. 5). Additional wells were stained with antibodies reactive with cytokeratin or vimentin to ensure cultures contained >90% trophoblasts.


Figure 5
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  		Figure 5: Photomicrographs demonstrating the proliferation of putative EVT progenitors isolated from an 8 week placenta and immunostained after 4 days in culture. Groups of isolated putative EVT progenitors stained with the proliferation marker Ki67 (A and B). That these cells were trophoblasts was confirmed by immunostaining with cytokeratin (C) and vimentin (D). As Ki67 is a nuclear stain cells stained with this antibody were not counterstained with haematoxylin, however those stained with antibodies against cytokeratin (C) and vimentin (D) were counterstained with haematoxylin

 
Putative EVT progenitors do not syncytialize over 4 days of culture
In order to determine whether the groups of putative EVT progenitors were syncytialising in culture, putative EVT progenitors cultured for 4 days were incubated with antibodies reactive with syncytiotrophoblast (G11) (Abumaree et al., 2006Go). This showed, isolated trophoblasts were negative for G11 on day 4 of culture (Fig. 6D). The Jar choriocarcinoma cell line which has previously been shown to express the G11 antigen was used as a positive control (Fig. 6H) (Abumaree et al., 2006Go). An irrelevant IgM class antibody (Jab-1) was used as a negative control and did not stain EVT outgrowths or isolated putative EVT progenitors. Additional wells were stained with antibodies reactive with cytokeratin or vimentin to ensure cultures contained >90% trophoblasts.


Figure 6
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  		Figure 6: Phase contrast photomicrographs demonstrating staining of putative EVT progenitors on day 5 of culture with antibodies reactive with (A) HLA-G; (B) class I MHC; (C) EVTs (Bo1D11 antibody) and (D) syncytiotrophoblast (G11 antibody). Sectioned first trimester placental tissue was used as a positive control for HLA-G (E) and class I MHC (F). EVT outgrowth from villous explants (G), developed with DAB were used as positive controls. The Jar choriocarcinoma cell line was used as a positive control for the G11 antibody (H)

 
A proportion of putative EVT progenitors differentiate into EVTs over 4 days in culture
In order to determine whether putative EVT progenitors were differentiating down the EVT lineage during culture, replicate wells of isolated putative EVT progenitors were immunostained with antibodies reactive with either class I MHC (W6/32), HLA-G or the EVT-specific marker Bo1D11 after 24, 48 or 96 h of culture. No expression of either HLA-G or class I MHC was observed on day 1 or 2 of culture. However, from day 3 of culture onwards a small proportion of putative EVT progenitors reacted with the Bo1D11, class I MHC and HLA-G specific antibodies. By 96 h of culture, EVT colonies all contained mixtures of HLA-G, class I MHC and Bo1D11 positive and negative cells with ~20% being HLA-G positive (Fig. 6).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
The lack of suitable human cell culture models in which cytotrophoblasts differentiate down the EVT lineage has hampered the understanding of the factors that control EVT differentiation and outgrowth. The identification of a population of cytotrophoblasts committed to EVT differentiation would therefore be an appealing target by which to establish such a model. Furthermore, if cytotrophoblasts in the first trimester are not one homogenous bipotent population, it is important to study the correct population of EVT progenitors to accurately understand the process of EVT outgrowth. Therefore, in this work the extended viability of the EVT progenitor population was used in order to isolate a population of putative EVT progenitors from first trimester placentae and to study them in greater detail.

The major lines of evidence in this work indicating that the putative EVT progenitors that have been isolated after extended explant culture are not bipotent are:

  1. Multilayered groups of EVT progenitors in villous tips were the only viable cells in the anchoring villi after 10 days of explant culture, and these did not induce regeneration of the surrounding syncytiotrophoblast.
  2. Isolated putative EVT progenitors stained strongly with antibodies reactive with {alpha}vbeta6 integrin. The {alpha}vbeta6 integrin is only expressed by villous cytotrophoblasts at sites of EVT column initiation, and not in monolayer cytotrophoblasts or other cell types in the villi (Zhou et al., 1997Go).
  3. The multilayered cytotrophoblasts in the cell islands at the tips of anchoring villi proliferate in order to drive the expansion of the EVT columns, and a high proportion stain with the proliferation marker Ki67, in comparison to the majority of monolayer villous cytotrophoblasts underlying the syncytiotrophoblast (Vivovac et al., 1995Go; Korhonen and Virtanen, 1997Go). After several days of culture, putative EVT progenitors were present in small colonies of actively proliferating cells, as shown by their expression of Ki67. This highlights the difference between these putative EVT progenitors, and those isolated from fresh first trimester placentae, which are predominantly monolayer villous cytotrophoblasts and do not proliferate in culture, but rather differentiate into syncytium (Morrish et al., 1997Go).
  4. Putative EVT progenitors did not syncytialize during 4 days of culture in vitro. Cytotrophoblasts isolated by traditional methods from fresh first trimester placentae undergo syncytialization within 1–2 days of culture (Morrish et al., 1997Go; Tarrade et al., 2001Go). In contrast, isolated EVT progenitors failed to stain with the syncytial marker G11, showing that they did not syncytialize over 4 days in culture. Since syncytialized trophoblasts do not proliferate (Boyd and Hamilton, 1970), the expression of Ki67 in these colonies of cells further confirms that the putative EVT progenitors did not syncytialize.
  5. A proportion of isolated putative EVT progenitors differentiated into EVTs. By the end of the 4 day culture period ~20% of cells stained with antibodies reactive with the EVT markers HLA-G, class I MHC and Bo1D11. These cells were sometimes observed adjacent to non-staining cells with trophoblast morphology on the plate. This suggests that a proportion of putative EVT progenitors were differentiating into EVTs. Furthermore, the existence of adjacent, non-staining cells suggests that many isolated EVT progenitors remain as undifferentiated progenitor cells for at least 4 days in culture.

We had also anticipated that the putative EVT progenitors might express tenascin, since it is present in anchoring villi immediately adjacent to sites of cytotrophoblast column initiation, but is not detected on monolayer villous cytotrophoblasts (Castellucci et al., 1991Go; Damsky et al., 1992Go). However, while putative EVT progenitors expressed the receptor for tenascin, {alpha}vbeta6 integrin, they did not stain with antibodies reactive with the extracellular matrix protein tenascin. It is not clear whether the tenascin in the proximity of EVT progenitors in vivo is expressed by these cells or by adjacent fibroblasts, possibly as a result of paracrine interactions with the EVT progenitors. Therefore, the lack of tenascin expression by putative EVT progenitors in vitro does not detract from the evidence that the putative EVT progenitors that have been isolated are likely to be the population identified as EVT progenitors in villous explants.

Our hypothesis that a distinct population of EVT progenitors exists in first trimester villi has support from the literature (Aboagye-Mathiesen et al., 1996Go; Tarrade et al., 2001Go; Nagamatsu et al., 2004aGo). Aboagye-Mathiesen et al. (1996)Go described the isolation of different populations of (i) mononuclear villous cytotrophoblasts that become committed to syncytium formation and (ii) a population of trophoblasts which appear in a ‘crazy pavement’ pattern and expressed markers of EVTs including {alpha}1 integrin, E-cadherin and class I MHC in vitro, from different stages of trypsinization (Aboagye-Mathiesen et al., 1996Go). Furthermore, a significant difference in cytotrophoblasts obtained from placentae of 10 weeks of gestation or less, or placentae >10 weeks of gestation has been observed (Aboagye-Mathiesen et al., 1996Go). In placentae of 10 weeks of gestation or less, trophoblasts with a flattened appearance that proliferated very slowly in culture were occasionally obtained and may have been similar to the putative EVT progenitor population described in this work (Aboagye-Mathiesen et al., 1996Go). In contrast, in placentae >10 weeks of gestation, even after 30 min of trypsinization, the majority of cells obtained were villous trophoblasts which had no ability to proliferate, and differentiated to form multinucleated syncytiotrophoblast in vitro (Aboagye-Mathiesen et al., 1996Go). The difference in the cytotrophoblast populations obtained at different gestational ages reported by Aboagye-Mathiesen et al., is in line with our previous work demonstrating that the proportion of cytotrophoblasts with the potential to form EVT outgrowth declines with increasing gestation (James et al., 2006Go), and supports the use of placentae only under 10 weeks of gestation in this study.

Using an adaptation of the method of Aboagye-Matheisen et al., Nagamatsu et al. (2004b)Go also isolated a population of cytotrophoblasts, from placentae of 10 weeks of gestation or less, that have similar properties to those we describe here (Nagamatsu et al., 2004aGo). In vitro, the isolated trophoblasts obtained by Nagamatsu et al. (2004a)Go, gradually upregulated {alpha}1 integrin, HLA-G and CD9, and did not syncytialize. Likewise, the putative EVT progenitors isolated in this work did not syncytialize, expressed CD9 after 3 days in culture, and a proportion of the cells also upregulated HLA-G after 2–3 days in culture.

One of the limitations in the isolation and culture of putative EVT progenitors at present is the low level (<10%) of fibroblast contamination observed in putative EVT progenitor isolates. Although the putative EVT progenitors proliferated in culture, their rate of proliferation was substantially less than that of the contaminating fibroblasts, and this limited the length of putative EVT progenitor cultures. Others have previously reported that CD9 can be used to immunodeplete fibroblasts from trophoblast isolates (Morrish et al., 1997Go; Verrijit et al., 1997Go). However, despite preliminary attempts, a CD9 immunodepletion step was not used to remove fibroblasts in cultures of isolated putative EVT progenitors for two reasons. First, the isolated putative EVT progenitors obtained expressed CD9 in vitro, and CD9 expression was observed in groups of cells in villous tips of fresh frozen first trimester sections, suggesting that CD9 may be expressed by the EVT progenitors in vivo. Second, the use of immunomagnetic beads appeared to substantially reduce the number of trophoblasts obtained at the end of the protocol, possibly due to the additional steps in the procedure affecting the viability of the cells being isolated, or as a result of a previously reported effect of non-specific adhesion of trophoblast cells to the immunomagnetic beads that results in a loss of some trophoblasts (Aboagye-Mathiesen et al., 1996Go). Despite this lack of specific fibroblast depletion, and even though the number of fibroblasts in villi are much greater than the number of EVT progenitors, the level of initial fibroblast contamination was extremely low, and the majority of cultures contained >90% pure trophoblasts 24 h after isolation. The contaminating fibroblasts were potentially derived from large core villi which were contained in our digests but which we have not examined for cellular viability (James et al., 2005Go).

One previous report has claimed to provide evidence that cytotrophoblasts are a bipotent population in the first trimester, however, this report has been disputed (James and Chamley, 2006Go). Taken together, the results we have presented here, and the results of Aboyage-Mathisen et al. (1996)Go and Nagamatsu et al. (2004aGo, bGo) strongly suggest that even in the first trimester not all villous cytotrophoblasts are identical and there appears to be at least two separate populations of villous cytotrophoblasts, one committed to syncytialization and the second committed to the EVT pathway.

The finding that villous cytotrophoblasts from first trimester placentae are not bipotent progenitors may explain why it is difficult to obtain large numbers of trophoblasts that either differentiate into an invasive EVT phenotype or proliferate following enzymatic digestion of fresh first trimester placentae, as the vast majority of villous cytotrophoblasts are contained in the villous monolayer and would be committed to the syncytiotrophoblast differentiation pathway. Therefore, results using cytotrophoblasts isolated from fresh first trimester placentae using the traditional ‘Kliman’ (Kliman et al., 1986Go) enzymatic digestion methods, should be interpreted with this in mind.

It has become evident that oxygen concentration has the ability to affect the differentiation of trophoblasts in the first trimester, and therefore physiologically relevant oxygen concentrations (1–3%) are recommended for the culture of first trimester trophoblasts (Caniggia and Winter, 2002Go; James et al., 2006Go). However, we have deliberately cultured the isolated putative EVT progenitors in 20% oxygen rather than low oxygen conditions for several reasons: (i) our previous study of EVT progenitor survival employed 20% oxygen (James et al., 2005Go); (ii) in order to microscopically examine cultures they would have had to be removed from their low oxygen environment, potentially inducing hyperoxic shock and (iii) in preliminary experiments (data not shown), the oxygen concentration did not appear to alter the behaviour of the putative EVT progenitors.

We have searched the literature extensively to try to find specific markers of the cytotrophoblasts in cell islands adjacent to trophoblast columns which we believe are EVT progenitors and in this work have examined all of the limited number of markers that have been published in this initial characterization of the isolated EVT progenitors. In future investigations, differential proteomic analysis may aid in the establishment of further markers specific for EVT progenitors. In this work, we have limited our initial characterization of the isolated EVT progenitors to single antibody staining for cytokeratin, vimentin, CD9, {alpha}vbeta6 integrin or Ki67. The in-depth characterization of this population of cells will require the use of double antibody staining to confirm the sequence and timing of differentiation events occuring in these cells.

In summary, we have isolated a population of putative EVT progenitor cytotrophoblasts, from first trimester placentae, that exhibit similar characteristics to those found in cell islands in villous tips in vivo. In addition, these cells did not syncytialize, and a proportion differentiated into EVT over 4 days in culture. This work provides further evidence to suggest that cytotrophoblasts in the first trimester are not a homogenous bipotent population.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
We wish to thank the staff at Epsom Day Unit, Greenlane Hospital, Auckland and Auckland Medical Aid Centre, Mt Eden, Auckland for their help in obtaining the tissue required for this study and the patients that donated their tissue for study. This research was funded by grants from the Evelyn Bond Trust and the University of Auckland Research Committee. J.J. is a recipient of a University of Auckland Postgraduate Scholarship.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on February 25, 2007; resubmitted on April 22, 2007; accepted on May 1, 2007.


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