Placenta 33 (2012) 88e93

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Placenta
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The contribution of apoptosis-inducing factor (AIF) to villous

trophoblast differentiation
M.R. Riddell a, b, B. Winkler-Lowen b, L.J. Guilbert b, *
a
Department of Physiology, 232 HMRC, University of Alberta, Edmonton, Canada T6G 2S2
b
Department of Medical Microbiology and Immunology, 625 HMRC, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Apoptosis is postulated to be a delayed but important part of the differentiation of placental villous
Accepted 15 November 2011 cytotrophoblasts (CT) into functional syncytiotrophoblast (ST). This hypothesis is based on the obser-
vation that the externalization of phosphatidylserine and the activation of caspase 8 are required for
Keywords: trophoblast differentiation. In contradiction to this hypothesis we have previously found that differen-
Pregnancy tiation occurs in the presence of both broad spectrum and caspase 8 specific inhibitors. Apoptosis-
Placenta
inducing factor (AIF) is a mitochondria-associated protein which is known to translocate to the
Villous trophoblast
nucleus and induce caspase-independent nuclear condensation, phosphatidylserine externalization and
Cytotrophoblast
Syncytiotrophoblast
cell death. Thus AIF nuclear translocation may result in the apoptotic-like features associated with
AIF trophoblast differentiation and may be an obligatory event for differentiation to proceed. AIF trans-
Apoptosis location was assessed in isolated primary trophoblasts by optical section microscopy of antibody stained
Differentiation cells. We found AIF to be strongly expressed in the villous trophoblast and that small amounts of AIF
AIF translocation were localized to the nucleus of the cells. Significantly, inhibitors of AIF translocation (calpeptin and zFA-
Trophoblast purification fmk) blocked translocation but not differentiation of the cells. We conclude that AIF translocation is not
involved in CT differentiation in isolated cell culture.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction cascade is a requirement for the differentiation and fusion of the CT

The syncytiotrophoblast (ST) is a large multinucleate cell in the
villous placenta. It forms the most external layer of the barrier
between the mother and fetus and is the site of nutrient, gas, and
waste exchange. The ST is non-proliferative and is maintained
exclusively through two processes of differentiation. Firstly, cyto-
trophoblast (CT) stem cells are stimulated to divide and the
daughter cells fuse with the over-lying syncytium. These fused CT
then differentiate to become a functional part of the ST [1]. A
second process of differentiation that occurs within the ST is the
packaging of aged nuclei into syncytial knots which are then
released into the maternal circulation [1,2].
The conditions required to stimulate CT differentiation remain
to be fully elucidated but it has previously been shown that CT
fusion requires caspase 8 protease activation and the externaliza-
tion of phosphatidylserine (PS) from the inner to the outer
membrane leaflet [3e10]. Jointly this work has been interpreted to
indicate that the initiation of the caspase-dependent apoptotic
* Corresponding author. Tel.: þ1 250 370 9742; fax: þ1 780 492 1308.
E-mail address: larry.guilbert@ualberta.ca (L.J. Guilbert).
[3,5,11]. The importance of caspase 8 activation in trophoblast
differentiation has been challenged in several publications where
trophoblast fusion and CT differentiation has been observed in the
presence of caspase 8 protease activity inhibitors [11,12].
The hypothesis linking trophoblast fusion and the initiation of
the apoptotic pathway relies heavily on anti-PS monoclonal
antibody-blocking experiments which showed PS externalization
to the outside of the plasma membrane is essential for trophoblast
fusion [10,27]. Aminophospholipids such as PS are normally
maintained in the inner leaflet of the plasma membrane and this
localization is actively maintained by aminophospholipid translo-
case [13]. PS externalization is a hallmark of early-apoptosis but is
also known to be associated with non-apoptotic cellular fusion in
myoblast to myotubule differentiation [14,15]. The cellular signals
that result in PS externalization in both apoptosis and myotubule
formation remain to be completely elucidated but mitochondrial-
mediated release of apoptogenic molecules such as apoptosis-
inducing factor (AIF) and cytochrome c are known to be involved
[16].
With evidence mounting that activation of the caspase cascade
may not be involved in the apoptotic-like features associated with
trophoblast differentiation we felt it a logical extension to examine

0143-4004/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.placenta.2011.11.008
 M.R. Riddell et al. / Placenta 33 (2012) 88e93
whether caspase-independent apoptosis may play a role in
trophoblast differentiation. Caspase-independent apoptosis is
mediated by the translocation of the mitochondrial flavoprotein
apoptosis-inducing factor (AIF) to the nucleus [17,18]. AIF is nor-
mally found in the mitochondrial intermembrane space and upon
apoptotic execution is released into the cytoplasm and translocates
to the nucleus where it causes chromatin condensation and
apoptosis. Caspase-independent apoptosis is also known to result
in PS externalization [16,19]. Cleavage of AIF from the mitochon-
drial inner membrane has been attributed to both cathepsin B and
calpain I and translocation has been effectively blocked using
chemical inhibitors of these proteases [20].
To test our hypothesis we utilized an in vitro model of isolated
primary CT cells stimulated to differentiate with the cell permeant
cAMP analog Br-cAMP to model trophoblast fusion and differenti-
ation. Although small amounts of AIF were localized to the nucleus
of half of CT 4 h after plating primary cells and stimulation with Br-
cAMP for 72 h increased the fraction of nuclei containing AIF to
approximately three quarters, blockade of AIF translocation to the
nucleus with calpain and cathepsin-B inhibitors did not lead to
a decrease in trophoblast fusion or hCG secretion. This data
supports a model of trophoblast differentiation that involves
neither AIF translocation to the nucleus nor caspase-independent
apoptosis.
2. Methods
2.1. Cells and tissues
Primary trophoblasts were isolated from normal term placentas at 39 weeks
gestation that were delivered by caesarean section without labor after patient
consent and full ethics review by the Alberta Health Services Ethics Committee,
Edmonton. The tissue arrived in the laboratory and isolation procedures were
started within 30 min of delivery. Purified villous cytotrophoblasts (CT) were iso-
lated by trypsin-DNAse digestion of minced chorionic tissue and immunoabsorbtion
onto immunoglobulin (Ig)-coated glass bead columns as previously described [21]
using antibodies to CD9 (clone 50H.19, house preparation), major histocompati-
bility complex (MHC) class I (clone W6/32, Harlan Sera-Lab, Crawley Down, Sussex,
UK) and MHC class II (clone 7H3, house preparation) then cryopreserved in fetal
bovine serum (FBS, GIBCO, Grand Island, NY) made 10% in dimethylsulfoxide
(DMSO) in liquid nitrogen. After plating onto tissue culture plastic, <0.01% of the
population were mesenchymal cells by vimentin staining as previously described
[22].
Villous tissue was prepared for sectioning immediately upon receipt of the
sample in the laboratory. Specifically, chorionic villi were dissected out and rinsed
three times in phosphate buffered saline containing 2% fetal bovine serum (Gibco,
Grand Island, NY) to remove maternal blood and the tissue was then cut into pieces
weighing w2 mg. A minimum of eight randomly selected pieces were fixed in 4%
paraformaldehyde for 1 h and then immersed in 30% sucrose (Sigma, Oakville, ON)
for 24 h. The tissue was then frozen in OCT (Sakura Tissue-Tekat, Torrance, CA)
at 70  C and 5 mm sections were cut on a Shandon tissue cryostat (Thermo Fisher
Scientific, Pittsburg, PA).
2.2. Cell and tissue culture
CT were rapidly thawed at 37  C, centrifuged, resuspended at 106 per ml in
Iscoves Modified Dulbecco’s Medium (IMDM, GIBCO) supplemented with 10% FBS
(IMDM-FBS) and antibiotics (end concentration penicillin 100 U/ml, streptomycin
100 mg/ml, Sigma, Oakville, ON) and plated as follows: One hundred mL were seeded
into 96 well tissue culture dishes (NUNC no. 167008, GIBCO) and incubated at 37  C
in 8% oxygen fully humidified and 5% CO2 atmosphere in a Forma model 3130
incubator (Marietta, OH) for 4 h. The plates were then washed twice with warm
IMDM and fed either 100 mL of pre-warmed (37  C) IMDM-FBS made 10 mM with the
cell permeable cAMP analog Br-cAMP (Sigma) or an equal volume of pre-warmed
IMDM-FBS (controls). The culture medium was changed every 2 days until har-
vested. Cultures were also carried out in 96 well glass-bottomed plates (VWR,
Mississauga, ON, Canada) for enhanced nuclear morphology resolution.
2.3. Measures of ST differentiation
Differentiation was assessed by cell fusion into syncytial units and by up-
regulation of hCG beta chain (hCG-b) secretion. Cell fusion was assessed by the
number of nuclei enclosed in desmoplakin-stained cellular boundaries [23] as
previously described [24]. Supernatants were assessed for hCG-b content with an
89
ELISA kit (DRG Instruments, Marburg, Germany) according to the manufacturer’s
instructions with standards provided as previously described [24]. Duplicate wells
were set up for every assessment.
2.4. AIF nuclear translocation
Experiments were carried out as specified in figure legends. Inhibitors of the
proteases (cathepsin-B and calpain) releasing AIF from the mitochondria [zFA-fmk
(Sigma), final concentration 20 mM and calpeptin (Calbiochem, La Jolla, CA), final
concentration 10 mM, respectively] were dissolved in DMSO and added to culture
medium at a dilution of 1/1000. This concentration of DMSO was also added to the
controls. Plated cells were fixed in 4% paraformaldehyde, permeabilized with 0.05%
Triton-X100, blocked for 1 h with 10% BSA, 20% goat serum and human IgG (627 mg/
mL; Sigma). Primary antibody against AIF or normal rabbit immunoglobulin were
then added, the plates incubated overnight at 4  C, followed by two washes in 0.1%
Tween-20 (PBST). Then Alexafluor goat anti-rabbit secondary antibody was applied
for 1hr at room temperature, followed by washes with PBST (0.1% Tween-20 in PBS)
and PBS (antibody concentrations given below). Nuclei were visualized with DAPI
(3 mM; Research Organics, Cleaveland) for 10 min at room temperature. Samples
were mounted with Vectashield (Vector Laboratories, Burlington, CA, USA) and
visualized with a Zeiss Axiovert 200 M microscope equipped with an ApoTome
optical slicer as described below. Nuclei were completely visualized with 0.4 mm
optical slices and considered negative if no AIF staining was seen in any slice.
2.5. Immunofluorescence staining, digital microscopy and image analysis
Antibodies for E-cadherin, desmoplakin and AIF were utilized for indirect
immunofluorescent staining of cultured isolated trophoblasts or fixed tissue
sections. Anti-AIF and anti-E-cadherin were applied to 4% paraformaldehyde fixed
tissue sections after antigen retrieval as detailed in [12]. Anti-AIF (3 mg/mL; R&D
Systems, Minneapolis), anti-E-cadherin (BD Transductions Laboratories, Franklin
Lake, NJ) and anti-desmoplakin (Sigma) antibodies or non-specific Ig controls
[mouse IgG2a, (R&D Systems); rabbit Ig (Santa Cruz);] were added at the same
concentrations and incubated at 4  C overnight as described previously [12]. After
washing with PBST Alexafluor goat anti-mouse and Alexafluor goat anti-rabbit
secondary was added as detailed above and nuclei were visualized with DAPI.
Unless otherwise stated, all images were obtained with an Olympus IX2-UCB
microscope equipped with a Roper Scientific camera and a Sutter Instruments
Lambda DG-4 fluorescent lamp (Olympus, Melville, NY). We used Slidebook 3.0
(Carsen, Markham, ON, Canada) as capture software and Image Pro-Plus (Media
Cybernetics; Del Mar, California) for analysis. Optical sections (0.4 mm) of anti-AIF
images employed a Zeiss Axiovert 200 M microscope equipped with an X-Cite 120
fluorescence illumination system with an ApoTome optical slicer module, an Axio-
Cam camera and was computer-controlled with AxioVision 4.5 (Zeiss, Goetingen,
Germany).
2.6. Annexin-V labeling
Trophoblasts were plated as for AIF translocation experiments above and were
treated upon thawing with staurosporine (0.3 mM) or Br-cAMP (10 mM) þ/ cal-
peptin (20 mM) or DMSO control. The cells were cultured for 24e72 h before washing
with Annexin V binding buffer and the addition of Annexin V-FITC (1:50 in Annexin
V binding buffer; Lonza, Walkersville, MD) for 1 h at 37  C. The cells were then again
washed with Annexin V binding buffer and fixed with 4% paraformaldehyde for
10 min at room temperature. After washing with PBS the nuclei were counterstained
with DAPI and visualized as in the immunofluorescent staining method above.
Triplicate wells of each treatment were plated for each of three independent
experiments using three placentas. The proportion of Annexin V positive cells were
counted and values from each of the three wells per treatment were averaged.
2.7. Statistics
All data came from minimally three sets of experimental data collected from
cells or tissues isolated from a minimum of three different placentas. One-way
analysis of variance (1-way ANOVA) with a Bonferroni post-hoc test were carried
out where applicable. Results were considered statistically significant if p < 0.05. The
statistical analysis was carried out using the statistics package in Prism software.
3. Results
3.1. Expression of AIF on trophoblasts in the villous placenta
AIF is commonly expressed in all cells [17,18] but the protein is
very strongly expressed in the villous trophoblast (Fig. 1) where the
ST and CT in the trophoblast are distinguished by E-cadherin
staining. CT are surrounded by E-cadherin and the ST, which
expresses only low levels, is not [25] but both express high levels of
90 M.R. Riddell et al. / Placenta 33 (2012) 88e93

Fig. 1. Expression of AIF and E-cadherin in the villous placenta. Three color indirect-immunofluorescence staining of AIF (red) and E-cadherin (green) and direct DAPI staining of
nuclei (blue) in a 22 wk placenta. Ai) AIF alone; Aii) High magnification image of selected CT and ST nuclei highlighted with a box from panel Ai; B) AIF (red), E-cadherin (green);
* ¼ E-cadherin-delineated CT. This photomicrograph is typical of >50 taken under the same conditions on three different placentas. Equal concentrations of isotype matched Ig did
not stain parallel sections.

AIF relative to the rather faint expression seen in the villous stroma.
AIF was highly expressed in isolated CT after 4 h of culture and was
predominantly localized to the mitochondria (Fig. 2). When optical
sections were taken of the entire nuclei of CT it was found that
small amounts of AIF were localized to the nucleus of w50% of CT
after 4 h. When CT were stimulated to differentiate into syncytium
with Br-cAMP for 3 days the proportion of nuclei containing small
amounts of AIF increased to w75%. Caspase-independent apoptosis
is associated with large-scale translocation of AIF to the nucleus in
multiple cell types [17,18] but small-scale translocation like that
observed with the isolated trophoblasts has not been characterized
in other models.
3.2. Inhibition of AIF translocation to the nucleus does not prevent
differentiation
Activation of AIF translocation from the mitochondria to the
nucleus can be blocked with the cathepsin and calpain inhibitors,
respectively zFA-fmk and calpeptin [20]. Thus, we next inhibited
translocation with inhibitors and determined whether they
blocked the small-scale AIF translocation observed in trophoblasts
as well as trophoblast fusion and hCG-b secretion. Calpeptin but
not zFA-fmk was able to significantly reduce AIF translocation to
the nucleus of trophoblasts (Fig. 3A) indicating that calpain I but
not cathepsin B is likely to be involved in the small-scale trans-
location of AIF in trophoblasts. Neither calpeptin nor zFA-fmk had
a significant impact on trophoblast fusion or hCG-b secretion,
though the effect of both of these agents may be masked by the
large variability in hCG-b secretion observed between individuals
(Fig. 3B and C). Thus it is unlikely that caspase-independent
apoptosis is involved in trophoblast differentiation. Calpeptin
treatment also failed to reduce the externalization of PS to the
outer membrane of trophoblasts (data not shown). Outer
membrane PS localization, as measured by Annexin V, did not
increase with time in culture or Br-cAMP stimulation of tropho-
blasts in primary cells and the proportion of Annexin V positive
cells was never more that w10% which we have previously found
to be the background level of apoptosis in cultured trophoblasts
(data not shown) [34].
4. Discussion
In this study, we used isolated cytotrophoblasts stimulated with
Br-cAMP as a model of villous cytotrophoblast differentiation. To
query whether caspase-independent apoptosis was involved in
trophoblast differentiation we examined the cellular localization of
the caspase-independent apoptotic mediator apoptosis-inducing
factor (AIF). Translocation of AIF from the mitochondria to the
nucleus is an essential step in caspase-independent apoptosis
[17,18]. We found that small amounts of AIF were within the nuclei
of a large proportion of CT and ST nuclei but that inhibition of this
translocation had no effect on differentiation. These results indicate
that caspase-independent apoptosis is unlikely to be involved in
trophoblast differentiation.
The hypothesis that cytotrophoblast fusion results from the
initiation of the classical caspase-dependent apoptotic cascade
(particularly caspase 8) is currently controversial in the field of
trophoblast differentiation [11,12,26]. We have previously pub-
lished using the same model of Br-cAMP-stimulated isolated
primary cytotrophoblast fusion used in this study and found that
both broad spectrum caspase inhibitors and caspase 8 specific
inhibitors were not capable of blocking trophoblast fusion [12].
 M.R. Riddell et al. / Placenta 33 (2012) 88e93 91

Fig. 2. AIF translocation from the cytoplasm to the nucleus in primary trophoblasts as a function of Br-cAMP-stimulated differentiation. Ai) AIF protein present (arrows) or not
present (arrowheads) in the nuclei of trophoblasts cultured for 4 h with medium alone. AIF is stained green, nuclei stained blue with DAPI, bar ¼ 25 mm for panels A and B. Isotype
antibody control staining for AIF was negative (data not shown); Aii) High magnification image of nuclei highlighted with a box from panel Ai where AIF translocation is present; B):
AIF protein in the cytoplasm, mitochondria and nucleus of cells incubated with Br-cAMP for 3 days. Bii) High magnification image of nuclei highlighted in panel Bi where AIF
translocation is present. C): Percent of nuclei with translocated AIF after culture with medium alone after 4 h and 3 days with Br-cAMP. Experiments were repeated in cells isolated
from three placentas. All experiments were carried out in an environment of 8% oxygen, 5% CO2.

Thus we were interested to examine whether apoptosis via the
caspase-independent mediator AIF could play a role in CT differ-
entiation. The hypothesis on the involvement of early-apoptosis in
trophoblast fusion also relies on data that shows that the exter-
nalization of PS is required for trophoblast fusion [11]. The mech-
anisms by which PS externalization occur in trophoblast
differentiation have previously been shown to be ATP and protein
kinase A (PKA)- dependent but independent of caspase activation
[27]. To strengthen our hypothesis that AIF was involved in
trophoblast differentiation the translocation of AIF to the nucleus
has been shown to temporally coincide with PS-efflux in other cell
systems [28]. We did not observe PS-efflux above basal apoptosis
levels in primary cells and did not see any changes in PS-efflux
when the cells were treated with calpeptin. These results lead us
to postulate that PS-efflux does not occur to the same degree as
previously observed in trophoblastic cell lines and that the small-
scale AIF translocation we observed is not involved in PS-efflux.
This hypothesis is currently being examined in detail and the
results will be presented elsewhere. In this communication we did
not find AIF nuclear translocation to be involved in trophoblast
differentiation. Instead, AIF was found in a high proportion of
trophoblast nuclei in small quantities throughout the differentia-
tion process.
This small-scale translocation of AIF to the nucleus is a novel
observation. In other cell systems and in vivo tissue where caspase-
independent apoptosis is occurring AIF nuclear translocation is
massive and is found throughout the nucleus [29,30]. We did not
observe massive translocation of AIF to the nucleus when primary
trophoblasts were stimulated with TNF-alpha and did not see any
increase in the proportion of nuclei containing small amounts of
AIF despite increases in the proportion of terminal deoxy-
nucleotidyl transferase dUTP nick end labeling (TUNEL) positive
nuclei (data not shown). This indicates that AIF translocation is also
not involved in TNF-alpha mediated apoptosis. Nucleus-filling
92 M.R. Riddell et al. / Placenta 33 (2012) 88e93
Fig. 3. Inhibition of AIF translocation fails to block trophoblast differentiation. A)
Percent of nuclei containing translocated AIF after treatment with Br-cAMP and
cathepsin-B inhibitor zFA-fmk or calpain inhibitor calpeptin. DMSO is the solvent
control for both inhibitors. B, C) Trophoblast fusion and hCG-b secretion was not
inhibited by zFA-fmk or calpeptin after 72 h of treatment with Br-cAMP. The histo-
grams in panels A through C are the average of 3 experiments with CT from 3 different
placentas. Bar graphs represent mean  SD. Panel C hCG-b secretion is normalized to
a medium control containing no Br-cAMP. * ¼ p < 0.05 versus control; all experiments
were carried out in an environment of 8% oxygen, 5% CO2.
translocation is what is classically associated with AIF-dependent
nuclear condensation and apoptosis, though AIF nuclear trans-
location is not observed under all conditions that stimulate
apoptosis and specific apoptotic signals appear to be required for
nuclear translocation [18,40]. Therefore other conditions that
stimulate apoptosis in trophoblasts may stimulate nucleus-filling
AIF translocation, but these remain to be described. It is currently
unknown why small amounts of AIF is found in trophoblast nuclei.
Br-cAMP treatment for 72 h increased the number of nuclei con-
taining low levels of AIF from approximately 50%e75% and co-
treatment with the calpain inhibitor calpeptin reversed the
increase (to 50%). Thus, it appears that at least a portion of the AIF
found in trophoblast nuclei relies on calpain cleavage of mito-
chondrial AIF.
AIF is not, to our knowledge, known to play any roles besides
stimulating nuclear condensation through an unknown mecha-
nism while localized to the nucleus. Mitochondrial AIF is involved
in stabilization of the electron transport chain and cells with
decreased expression of AIF are more susceptible to oxidative stress
[31]. It is additionally thought to play a major role in mitochondrial
metabolism: tissue specific deletion of AIF in muscle or liver results
in increased glycolytic rates, insulin hypersensitivity and a resis-
tance to diabetes [31]. So though the lethal functions of AIF do not
seem to be active during trophoblast differentiation the vital
functions of the protein are likely to be important for trophoblast
survival. The small amount of nuclear AIF observed in trophoblasts
may somehow play a role in cellular metabolism or regulation of
trophoblast oxidative stress.
Membrane fusion events have been extensively studied in the
area of fusion of enveloped viruses with host cells but the molec-
ular mechanisms involved in cellecell membrane fusion are
considerably less well characterized. Studies of macrophage and
myoblast fusion are likely to bring light to common cellular
mechanisms involved in trophoblast fusion. Cellular fusion is
undoubtedly a highly regulated process, no matter the cell type
involved, with three general steps likely common: (1) cells must
acquire the capacity to fuse in order to prevent random fusion
events; (2) fusion competent cells must bring their membranes
within close contact and; (3) the unification of the membranes and
rearrangement of the cellular components. Almost all of our current
knowledge of trophoblast differentiation examines aspects of the
first two steps of cellular fusion. Multiple stimuli such as growth
factors, cytokines, and hormones have been shown through in vitro
work to induce trophoblast fusion and are involved in the estab-
lishment of a fusion competent state in CT. Cellular signaling
pathways from these multiple signals stimulate the expression of
different transcription factors, such as GCM1, which has been
shown to control the balance between trophoblast proliferation
and fusion [24,33]. These signaling cascades also result in the
differential expression pattern of multiple membrane constituents,
such as syncytin-1, syncytin-2, E-cadherin, cadherin-11, CD98, and
zona occudens 1 which then allow close contact in order for fusion
to progress [32,33,35e37]. Additionally the involvement of full
length caspase 8 in a non-apoptotic capacity is implicated in cas-
pase 8 siRNA experiments [3,11,12,26].
Recently several molecules have been identified to be involved
in both macrophage and myoblast fusion [38]. Specifically the
GTPase Rac1 and guanine-nucleotide exchange factor Dock 180
play an important roles in fusion of both cell types, therefore
exploration of these signaling molecules and pathways deserve
attention in the trophoblast [39]. The involvement of PS external-
ization and the mechanisms involved in this apparently conserved
event of cellular fusion require additional attention in the tropho-
blast field. Examination of additional cell signaling cascades and
the cellular machinery involved in PS externalization during
 M.R. Riddell et al. / Placenta 33 (2012) 88e93
cellecell fusion compared to those utilized in apoptosis has broad
applications across fusion competent cell types and is currently
being examined in our laboratory.
When the results of this study and those of previous studies
which have found that caspase activation is not required for
trophoblast fusion are considered together it becomes apparent
that trophoblast fusion in vitro does not involve either known
apoptotic cascade. Therefore trophoblast differentiation occurs via
an apoptosis-independent mechanism, as has been found for both
macrophage and myoblast fusion. Therefore future research should
focus on elucidating the non-apoptotic signals contributing to
differentiation.
Acknowledgments
We would like to thank our research nurse Donna Dawson for
the collection of all of our placental samples. We also wish to thank
the Canadian Institutes of Health Research for their support of this
project (grant number MOP-82892) and for the CIHR Strategic
Training Program in Maternal-Fetal-Newborn Health which partly
supported one of us (MR).
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