feasibility and potential of in utero foetal membrane-derived cell transplantation
TRANSCRIPT
ORIGINAL PAPER
Feasibility and potential of in utero foetal membrane-derived cell transplantation
Maddalena Caruso • Patrizia Bonassi Signoroni • Roberto Zanini •
Lorenzo Ressel • Elsa Vertua • Piero Bonelli • Maria Dattena •
Maria Vittoria Varoni • Georg Wengler • Ornella Parolini
Received: 25 July 2013 / Accepted: 9 October 2013
� Springer Science+Business Media Dordrecht 2013
Abstract Cells isolated from foetal membranes of
human term placenta display multiple properties,
including some features of stem/progenitor cells,
together with immunomodulatory actions and the ability
to secrete bioactive soluble factors. Whilst such prop-
erties support the potential applicability of these cells in
transplantation settings aimed at regenerating/repairing
tissues in adults, theoretically, using these cells in
prenatal treatment strategies may also be achievable. To
assess the feasibility of a foetal membrane-derived cell-
based therapeutic treatment during foetal development,
we firstly addressed the question of whether in utero
transplantation using these cells was possible. To this
end, we assessed postnatal microchimerism after trans-
plantation of amniotic membrane-derived cells (a
mixture of both mesenchymal stromal/stem cells and
epithelial cells) in foetal sheep. Transplantation was
performed with or without human umbilical cord blood
mononuclear cells and chorionic membrane-derived
mesenchymal stromal/stem cells, and was followed by a
postnatal booster cell injection. Lambs were euthanized
2–4 months postnatally and their organs/tissues were
analysed for microchimerism through detection of
human DNA. Human DNA was found in almost all
tissues of all of the lambs, with the seemingly random
appearance of human cells in some of the analysed
tissues suggesting long-term human microchimerism
and donor cell migration after in utero/postnatal booster
xenotransplation. Differences in microchimerism tissue
distribution between animals transplanted with different
cell types are discussed. This pilot study adds to ongoing
efforts by different investigators to explore the potential
M. Caruso � P. Bonassi Signoroni � L. Ressel �E. Vertua � G. Wengler � O. Parolini (&)
Centro di Ricerca E. Menni, Fondazione Poliambulanza-
Istituto Ospedaliero, Via Bissolati 57, 25124 Brescia, Italy
e-mail: [email protected];
R. Zanini
U.O. Ostetricia e Ginecologia, Fondazione
Poliambulanza-Istituto Ospedaliero, Via Bissolati 57,
25124 Brescia, Italy
L. Ressel
School of Veterinary Science, University of Liverpool,
Leahurst Campus, Chester High Road, Neston CH64 7TE,
UK
P. Bonelli � M. Dattena
Dipartimento per la Ricerca nelle Produzioni Animali,
AGRIS Sardegna, Loc. Bonassai, 07040 Olmedo, SS,
Italy
M. V. Varoni
Dipartimento di Medicina Veterinaria, Universita di
Sassari, Via Vienna 2, 07100 Sassari, Italy
123
Cell Tissue Bank
DOI 10.1007/s10561-013-9402-0
of in utero cellular transplantation, and warrants further
investigation of using foetal membrane-derived cells
for prenatal cell therapies.
Keywords In utero transplantation � Human
placenta � Foetal membrane-derived stem/
progenitor cells � Human umbilical cord blood
mononuclear cells � Foetal sheep � Human
microchimerism
Abbreviations
IUT In utero stem/progenitor cell transplantation
MSCs Mesenchymal stromal/stem cells
HSCs Hematopoietic stem/progenitor cells
CBMCs Human umbilical cord blood mononuclear
cells
hAMSCs Human amniotic mesenchymal stromal/
stem cells
hCMSCs Human chorionic mesenchymal stromal/
stem cells
hAECs Human amniotic epithelial cells
CISH Chromogenic in-situ hybridization
hALU Human ALU repeats
Introduction
In utero stem/progenitor cell transplantation (IUT) is
an attractive therapeutic approach for treating prena-
tally diagnosable genetic/developmental diseases
which are amenable to cell transplantation (Flake
2004; Muench 2005; Tiblad and Westgren 2008;
Pixley and Zanjani 2013).
Results from animal studies, mainly after trans-
plantation of hematopoietic stem/progenitor cells
(HSCs), but also other cell types, including mesen-
chymal stromal/stem cells (MSCs), support IUT’s
feasibility both in allogeneic and xenogeneic settings
(Flake 2004; Liechty et al. 2000; Mackenzie and Flake
2001; Muench 2005; Schoeberlein et al. 2005; Tiblad
and Westgren 2008). However, the full clinical
potential of IUT in humans remains to be reached,
with positive results currently limited to cases of
immunodeficiencies (Wengler et al. 1996; Flake et al.
1996; Flake 2004; Muench 2005; Tiblad and Westgren
2008).
A major obstacle to success of IUT is low/
inadequate chimerism/engraftment levels achieved
(Flake 2004; Muench 2005; Tiblad and Westgren
2008; Pixley and Zanjani 2013). Maximizing cell
dosage and serial cell administrations, including
postnatal booster cell injections, have been shown to
improve donor cell engraftment (Milner et al. 1999;
Pixley and Zanjani 2013). Meanwhile, co-transplan-
tation of bone marrow-derived stromal cell progeni-
tors with HSCs has also been shown to increase HSC
engraftment (Almeida-Porada et al. 1999; Almeida-
Porada et al. 2000).
A variety of donor cell sources have been used for
IUT (Muench 2005). Here we considered two easily
procured and ethically sound human sources: umbil-
ical cord blood and term placenta. While IUT using
umbilical cord blood-derived cells is relatively well-
documented (e.g. Young et al. 2003; Noia et al. 2003;
Wengler et al. 2005; Abellaneda et al. 2012), literature
regarding IUT using placenta-derived cells is some-
what scant.
Placental tissues, both of foetal and maternal origin,
harbour cells with several interesting properties,
including expression of stem cell/pluripotency mark-
ers and of different lineage-associated markers (Bailo
et al. 2004; In‘t Anker et al. 2004; Parolini et al. 2008;
Manuelpillai et al. 2011), demonstrated ability to
differentiate in vitro towards cells of multiple lineages
(Soncini et al. 2007; Miki et al. 2005; Ilancheran et al.
2007; Parolini et al. 2008; Manuelpillai et al. 2011),
and the potential, although debated, to also undergo
multilineage differentiation in vivo (Moodley et al.
2010; Marongiu et al. 2011). These cells also display
immunomodulatory abilities in vitro (e.g. Bailo et al.
2004; Wolbank et al. 2007; Li et al. 2007; Banas et al.
2008; Magatti et al. 2008; Magatti et al. 2009;
Vellasamy et al. 2012), and maintain their ability to
suppress mitogen-induced peripheral blood mononu-
clear cell (PBMC) proliferation (Tee et al. 2013) and
the expression of immune-related/immunomodulatory
molecules after differentiation towards cells of differ-
ent types in vitro (Tee et al. 2013; La Rocca et al.
2013). Taken together, all of these properties, along
with the ability to secrete several bioactive and trophic
soluble factors (Silini et al. 2013), suggest the
potential applicability of placental cells for regener-
ation/repair of tissues in adults, as indeed supported by
different pre-clinical studies (Parolini and Caruso
2011).
Theoretically, these properties of placental cells
could also make them useful for prenatal cell therapy.
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123
Exploration of this hypothesis by others, using
placental cells isolated from species other than
humans, has led to the demonstration that rat amniotic
membrane-derived cells collected during the embry-
onic stage could be transplanted into the rat foetal
brain through transuterine intraventricular injection
(Marcus et al. 2008). Even in the absence of neuronal
differentiation, donor cells were able to survive in
cortical and vascular regions up to two and a half
months postnatally, which was the longest time point
of examination, with no evidence of immunological
rejection or tumour formation (Marcus et al. 2008).
Meanwhile, transplantation of adenoviral AdlacZ-
transfected foetal rat amniotic epithelial cells into the
foetal rat liver by in utero manipulation has been
shown to result in the persistence of X-gal-positive
cells for up to 2 weeks after birth (Takahashi et al.
2002). If we instead consider cells isolated from
human placenta, studies to date have shown that after
IUT, MSCs from human central placental cotyledons
engraft multiple rat tissues, even though at low levels,
and persist for 12 weeks postnatally, with some
indication of specific lineage differentiation after
transplantation (Chen et al. 2009). Some evidence of
the presence of human placental piece-derived MSCs
in foetal murine organs after their intrauterine trans-
plantation in mice was also observed (Groza et al.
2012). Meanwhile, human amniotic epithelial cells
have been shown to attenuate inflammation-induced
foetal lung injury in sheep, either when they were
delivered through the foetal intravenous or intratra-
cheal route, or both (Vosdoganes et al. 2011), and have
also been shown to mitigate ventilation-induced
preterm lung injury and differentiate into alveolar
cells in foetal sheep (Hodges et al. 2012). However, in
both of these two studies, post birth human micro-
chimerism was not investigated.
Our group, together with others, has previously
reported several observations regarding the valuable
properties of amniotic epithelial cells and MSCs
isolated from human foetal membranes (amniotic
membrane and chorionic membrane) of term placenta,
including the fact that these cells can successfully
long-term engraft newborn animals (Bailo et al. 2004;
Soncini et al. 2007; Parolini et al. 2008; Cargnoni et al.
2009; Manuelpillai et al. 2011; Cargnoni et al. 2012).
However, the possibility of using these cells for IUT in
large animal models and achieving long-term micro-
chimerism after birth remains largely unexplored.
Here we report the results of our preliminary efforts
to address the feasibility of IUT with human foetal
membrane-derived cells and umbilical cord blood
mononuclear cells (CBMCs), separately or through
co-transplantation, in the well-established foetal sheep
model, maximizing cell dosage through multiple cell
transplantation (two IUT and one postnatally), and
assessing human microchimerism up to 4 months after
birth.
Materials and methods
Ethics statements
Human umbilical cord blood (n = 20) and human
term placentas (n = 20) were collected after maternal
informed consent and according to the guidelines of
the Ethical Committee of the Catholic Hospital
(CEIOC) and of the Ethical Committee of the hospital
Fondazione Poliambulanza-Istituto Ospedaliero (Bre-
scia, Italy).
Animal experiments were carried out in accordance
with current Italian legislation (D.L. 116/92).
Isolation and preparation of cells
for transplantation
Human donor cells used for transplantation were
obtained as follows:
• CBMCs were isolated from heparinized umbilical
cord blood by lymphoprep density gradient centri-
fugation (Axis-Shield, Oslo, Norway), according to
the manufacturer’s instructions. CBMCs were col-
lected from the interface and washed twice in
phosphate-buffered saline (PBS; Sigma, St Louis,
MO, USA). All recovered cells were CD45-positive
as demonstrated by flow cytometry analysis.
• Foetal membrane-derived cells, including amni-
otic membrane-derived cells [amniotic mesenchy-
mal stromal/stem cells (hAMSCs) and amniotic
epithelial cells (hAECs)] and chorionic mem-
brane-derived cells [chorionic mesenchymal stro-
mal/stem cells (hCMSCs)], were isolated as
previously described (Soncini et al. 2007). The
phenotype of hAMSCs and hCMSCs was as
previously reported (Soncini et al. 2007; Parolini
et al. 2008; Magatti et al. 2012). All collected
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123
hAECs were positive for CD166 expression as
demonstrated by flow cytometry analysis.
All of the above described cell types were cryo-
preserved immediately after isolation, in 10 % DMSO
supplemented with 90 % foetal bovine serum (Sigma),
and were kept frozen until their use in transplantation
procedures, at which point they were resuspended in
PBS supplemented with 100 U/ml penicillin and
100 lg/ml streptomycin (P/S, both from Euroclone,
Whetherby, UK).
Cell transplantation, experimental groups
and sample collection
The overall experimental design, including the trans-
plantation and sampling schedule, is depicted in
Fig. 1. In detail:
• IUT was performed intraperitoneally in n = 16
pregnant ewes at day 55–60 and once again at day
95–105 of gestation, by injecting cells into
recipient foetuses (n = 24) through an ultra-
sound-guided injection system without gravid
uterus exposure (Wengler et al. 2005; Young
et al. 2003). A further intravenous cell injection
was performed at 1 day post birth. Each of these
three cell injections was performed with the same
total number of cells, as specified below.
• foetuses were randomly assigned to three exper-
imental groups: group I, foetuses (n = 8) trans-
planted with 2.0 9 107 amniotic membrane-
derived cells (hAMSCs ? hAECs) for each cell
injection, at an approximate ratio of hAECs:
hAMSCs of 10:1; group II, foetuses (n = 6)
transplanted with 2.0 9 107 CBMCs for each
cell injection; group III, foetuses (n = 10)
Fig. 1 Experimental design
and groups; transplantation
and sampling schedule. IUT
was performed at day 55–60
(transplantation 1) and at
day 95–105 (transplantation
2) of gestation, and was
followed by a further
intravenous cell injection at
1 day post birth
(transplantation 3). Foetuses
were randomly assigned to
the three experimental
groups indicated in the
figure, which also reports the
type and amount of cells
used for each of the three
cell injections. Peripheral
blood samples (PB) were
collected at\20 days and
once again between 20 and
50 days after birth. The
lambs were euthanized
between 70 and 120 days
after birth and sampling of
tissues was performed for
subsequent PCR and/or in-
situ hybridization analyses
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123
co-transplanted with a total of 2.5 9 107 foetal
membrane-derived cells (hAMSCs ? hAE-
Cs ? hCMSCs) and CBMCs (60 % foetal mem-
brane cells ? 40 % CBMCs), for each cell
injection. The injection volume was 1 ml PBS
plus P/S.
• Peripheral blood samples were collected at
\20 days and once again between 20 and 50 days
after birth from each lamb under aseptic conditions
and following standard procedures. The lambs
were then euthanized between 70 and 120 days
after birth and a widespread sampling of tissues
was performed. All samples collected were frozen
at -80 �C and some samples were formalin-fixed
for subsequent polymerase chain reaction (PCR)
and/or in-situ hybridization analyses, respectively.
Microchimerism analysis by PCR
Total DNA was extracted from all frozen tissue
samples using the MagAttract DNA Mini M48 Kit and
the Bio robot M48 robotic workstation (both from
Qiagen, Hilden, Germany), according to the manu-
facturer’s instructions. The integrity and quality of the
isolated DNA were evaluated by electrophoresis on a
1 % agarose gel and ethidium bromide staining. To
assess the presence of human DNA sequences, DNA
samples were analyzed by PCR using specific primers
for the human cytochrome B mitochondrial gene, as
previously described (Soncini et al. 2006).
Chromogenic in-situ hybridization
Four-lm-thick formalin-fixed paraffin-embedded tis-
sue sections (two sections for each biopsy, with an
inter-section distance of approximately 100 microns)
from each of 46 randomly chosen biopsies harvested
from 10 lambs with PCR-positive organs/tissues (3
from group II, and 7 from group III), were subjected to
chromogenic in-situ hybridization (CISH) to detect
human ALU repeats (hALU), using a digoxigenin-
labelled hALU probe (Space SRL, Italy) and the Spot-
light CISH Polymer detection kit (Invitrogen, CA,
USA), according to the manufacturer’s instructions.
Sections from term human placenta were used as
positive controls for the CISH technique.
Results and discussion
All intrauterine transplanted foetuses were carried to
term. The absence of foetal loss compares favourably
with previous reports using this same ultrasound-
guided cell injection system (Young et al. 2003), which
is less invasive and traumatic than IUT techniques
requiring uterus exposure (Zanjani et al. 1992; Oppen-
heim et al. 2001). Three lambs of group I, and one lamb
of groups II and III died soon after delivery due to
labour complications which were not clinically related
to the transplantation procedure. Therefore, a total of
19 lambs received the postnatal cell injection and were
finally available for chimerism analysis.
All 19 transplanted animals showed normal growth
rates and no evident clinical symptoms of rejection or
graft versus host disease.
Shortly after birth, all experimental groups
included animals whose peripheral blood was PCR-
positive for human DNA (Table 1). Consistent with
previous findings (Almeida-Porada et al. 2000), the
fraction of the total of the analysed animals with
chimeric blood decreased in each group with time after
transplantation (Table 1).
All 19 euthanized lambs showed human micro-
chimerism, as demonstrated by PCR analysis, for up to
4 months after birth and in almost all organs/tissues,
suggesting that human transplanted cells were capable
of widespread migration after IUT/postnatal booster
xenotransplantation (Table 2).
Interestingly, in animals transplanted with amniotic
membrane-derived cells alone (group I), donor DNA
was found in different organs, including brain and
lung, where we previously reported human micro-
chimerism after transplantation of foetal membrane-
derived cells in newborn animals (Bailo et al. 2004).
Comparison of microchimerism organ/tissue dis-
tribution between experimental groups revealed some
notable differences. For example, when animals were
transplanted with amniotic membrane-derived cells
alone (group I), human DNA was not detected in the
thymus and only 1/5 of animals had chimeric bone
marrow and spleen (Table 2). Meanwhile, the number
of animals with chimeric bone marrow was higher in
the group that received CBMCs with foetal mem-
brane-derived cells (group III) than in animals trans-
planted with CBMCs alone (group II) (Table 2).
These findings suggest that amniotic membrane-
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123
derived cells alone do not display a preferred seeding
for hematopoietic tissues, and also that, consistent
with previous reports (Almeida-Porada et al. 2000),
the presence of MSCs (as in group III which includes
animals transplanted with a mixture of amniotic and
chorionic membrane-derived MSCs, along with
CBMCs and hAECs) may favour CBMC (HSC) bone
marrow engraftment, even though further study is
paramount to understand which of the transplanted
cell populations are actually present in the host tissues,
and the level of microchimerism/engraftment reached.
To gain some additional information on the human
microchimerism achieved, some randomly chosen
tissues of PCR-positive lambs were also analysed by
an in-situ hybridization technique for the presence of
human ALU repeats (hALU). While far from allowing
us to claim to provide comprehensive results, this
analysis does, however, provide some interesting
indications for further investigation. For example,
we detected positively hALU-stained cells in the heart
and in the brain tissues, as single cells or in clusters
(Fig. 2), in line with the fact that we found PCR-
positive signal in these organs/tissues (Table 2), and
also in accordance with reports from other authors
who detected positive donor cell staining in the brain
after intraperitoneal IUT in sheep foetuses, through
postnatal evaluation (Liechty et al. 2000; Mackenzie
and Flake 2001), or by more restricted analyses of the
transplanted foetuses before birth (Schoeberlein et al.
2005). Further characterization of these hALU-
positive cells, in terms of both human gene expression
and functions, will undoubtedly provide additional
evidences of the apparent presence of human cells
integrated within the sheep tissue background. The
fact that hALU-positive cells were found in clusters in
the brain also warrants more extensive investigation of
whether this may be an indication of transplanted cell
proliferation and/or if the observed cluster formation
is in some way related to the ability of donor cells to
access and migrate through brain tissues/regions, as
suggested for neural precursors (Brustle et al. 1997),
bone marrow stromal cells (Munoz-Elias et al. 2004)
and rat amniotic membrane cells (Marcus et al. 2008)
after IUT into the developing rodent brain.
In terms of the vision to develop a future therapeu-
tic application, it is however a promising sign that in
all analysed samples, host tissue surrounding inte-
grated putative human cells was morphologically
normal, without signs of inflammatory infiltration,
organ degeneration or necrosis.
In conclusion, this pilot study confirms the possi-
bility of achieving postnatal microchimerism after
transplantation of CBMCs into sheep foetuses, and is
also the first study to support this achievability for
human foetal membrane-derived cells, either alone or
Table 1 Human microchimerism detection by PCR in
peripheral blood samples
Sampling time
after birth
Group I
(n = 5)
Group II
(n = 5)
Group III
(n = 9)
Positive/tot Positive/tot Positive/tot
\20 days 4/5 5/5 8/9
20\ day \50 2/5 2/5 4/9
For each experimental group, the fraction of the total of the
analysed animals with PCR-positive signals in the peripheral
blood (Positive/tot) is indicated
Table 2 Human microchimerism detection by PCR in lamb
organs/tissues
Organs Group I
(n = 5)
Group II
(n = 5)
Group III
(n = 9)
Positive/
tot
nd Positive/
tot
nd Positive/
tot
nd
Lymph node 3/5 – 4/5 – 4/9 –
Spleen 1/5 – 1/5 – 7/9 –
Thymus 0/5 – 4/5 – 7/9 –
Bone marrow 1/5 – 2/5 – 5/7 2
Kidney 3/5 – 0/5 – 6/9 –
Liver 1/5 – 1/5 – 6/9 –
Omentum – 5 4/5 – 8/9 –
Heart 3/5 – 4/5 – 7/9 –
Aorta – 5 5/5 – 8/9 –
Lung 2/5 – 4/5 – 7/9 –
Muscle leg 3/5 – 4/5 – 7/8 1
Diaphragm – 5 4/5 – 9/9 –
Brain 3/5 – 4/5 – 7/9 –
Cerebellum – 5 5/5 – 9/9 –
Spinal cord – 5 5/5 – 7/9 –
Medulla
oblongata
– 5 3/5 – 5/9 –
Organ/tissue distribution of human DNA as determined by
PCR in animals of the three experimental groups. Positive/tot
indicates the fraction of the total number of analysed animals
with PCR-positive signals in a specific tissue. nd (not
determined) indicates the number of animals which were not
analysed in each group
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when co-transplanted with CBMCs. It also suggests a
variable pattern of organ/tissue microchimerism
depending on the cell type transplanted, interestingly
showing higher chimerism in bone marrow and
thymus when CBMCs were co-transplanted with
foetal membrane-derived cells. The fact that in this
study we did not observe evident signs of rejections
supports the hypothesis that this transplantation
method may represent a strategy for inducing immune
tolerance to transplanted cells, although this aspect
requires further investigation. Furthermore during the
life span of these animals of up to 4 months and also
after multiple cell injections, we did not observe any
tumour formation within the tissues and organs
analysed.
Conceived as a preliminary study, this work
inevitably leaves a number of open questions. For
example, as already mentioned above, while the level
of human/sheep chimerism achieved remains to be
determined, perhaps even more important information
could be gained through further investigation of the
presence of human cells integrated in the host tissues.
Provided that these are indeed human cells and not the
result of a transfer of nucleic acid sequences from
donor cells to host cells, further studies are warranted
to explore, for example, whether transplanted cells
retain their identity in the host tissues or if, in response
to host microenvironmental cues, they shift their gene
expression and/or their secretory ability toward a
specific cell lineage and/or toward a particular para-
crine action. The elucidation of these aspects will also
provide additional elements toward our understanding
of whether integrated human cells may benefit prena-
tal cell therapy by virtue of the transplanted cells’
capacity to undergo site-specific differentiation after
transplantation or if instead, as is becoming increas-
ingly favoured through transplantation studies of these
cells in adults, due to the paracrine factors these cells
release, or finally, whether both of these processes are
at play. Furthermore, studies applying IUT with
placental cells into models displaying tissue/organ
pathology will undoubtedly yield interesting data
toward further elucidation of the homing of trans-
planted cells.
Fig. 2 Chromogenic in-situ hybridization for human ALU
repeats in selected tissues from transplanted lambs. a Sections
from term human placenta were used as positive controls for the
in-situ hybridization technique. Nuclei positive for human ALU
repeats (hALU) were detected throughout the placental section.
The asterisk indicates the zone of the boxed magnification (scale
bar 100 microns) corresponding to the amniotic membrane:
hALU-positive nuclei were found both in the epithelial (arrow)
and mesenchymal (arrowhead) layers. b Representative micro-
photograph of a heart section from a lamb from group III with
PCR-positive tissue samples. Heart sections from one of three
analysed lambs of group III were hALU-positive. A hALU-
positive cell (arrow) was detectable and surrounded by
morphologically normal, hALU-negative myocardial sheep
cells (arrowhead), (scale bar 25 microns). c Representative
microphotograph of a brain section from a lamb from group III
with PCR-positive tissue samples. Brain sections from four of
five analysed lambs of group III were hALU-positive. A cluster-
like structure (arrow) of hALU-positive cells was detectable and
surrounded by normal hALU-negative sheep neurons (arrow-
head), (scale bar 25 microns)
b
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In line with others who have set out to explore the
potential of prenatal cell therapy, overall this study
provides data which warrant further efforts to define
whether and what type of foetal cells might have
therapeutic potential when applied during foetal
development.
Acknowledgments The authors thank the physicians and
midwives of the Department of Obstetrics and Gynaecology of
Fondazione Poliambulanza-Istituto Ospedaliero, Brescia, Italy
and all of the mothers who donated placenta and umbilical cord
blood. We sincerely thank also Dr. Marco Evangelista for help
in editing the manuscript. This work has been supported by
Regione Autonoma Sardegna (RAS) L.R. 7/2007 Bando 2008.
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