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REPRODUCTION
RESEARCH
Isolation, characterization and differentiation of mesenchymal
stem cells from amniotic fluid, umbilical cord blood and
Wharton’s jelly in the horse
Eleonora Iacono, Lara Brunori, Alessandro Pirrone, Pasquale Paolo Pagliaro
1
, Francesca Ricci
1
,
Pier Luigi Tazzari
1
and Barbara Merlo
Department of Veterinary Medical Sciences, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia,
Bologna, Italy and
1
Immunohaematology and Transfusion Center, Policlinico S.Orsola-Malpighi, Bologna, Italy
Correspondence should be addressed to E Iacono; Email: eleonora.iacono2@unibo.it
Abstract
Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbilical cord blood (UCB) and amnion.
This work aimed to identify and characterize stem cells from equine amniotic fluid (AF), CB and Wharton’s Jelly (WJ). Samples were
obtained from 13 mares at labour. AF and CB cells were isolated by centrifugation, while WJ was prepared by incubating with an
enzymatic solution for 2 h. All cell lines were cultured in DMEM/TCM199 plus fetal bovine serum. Fibroblast-like cells were observed in
7/10 (70%) AF, 6/8 (75%) CB and 8/12 (66.7%) WJ samples. Statistically significant differences were found between cell-doubling times
(DTs): cells isolated from WJ expanded more rapidly (2.0G0.6 days) than those isolated from CB (2.6G1.3 days) and AF (2.3G1.0 days)
(P!0.05). Positive von Kossa and Alizarin Red S staining confirmed osteogenesis. Alcian Blue staining of matrix glycosaminoglycans
illustrated chondrogenesis and positive Oil Red O lipid droplets staining suggested adipogenesis. All cell lines isolated were positive for
CD90, CD44, CD105; and negative for CD34, CD14 and CD45. These findings suggest that equine MSCs from AF, UCB and WJ appeared
to be a readily obtainable and highly proliferative cell lines from a uninvasive source that may represent a good model system for stem cell
biology and cellular therapy applications in horses. However, to assess their use as an allogenic cell source, further studies are needed for
evaluating the expression of markers related to cell immunogenicity.
Reproduction (2012) 143 455–468
Introduction
Over the past decade, stem cell research has emerged
as an area of major interest due to its potential in
regenerative medicine applications (Stocum 2001). The
three alternative stem cells are embryonic stem cells
(ESCs), adult stem cells (ASCs) and induced pluripotent
stem (IPS) cells.
ESCs are derived from blastocysts that propagate
readily (Thomson et al. 1998) and are capable of forming
embryoid bodies that generate a variety of specialized
cells including neural, cardiac and pancreatic cells
(Reubinoff et al. 2000). The establishment of horse ESC
lines is of interest for creation of models of human
genetic diseases and cell transplantation therapies.
Horse ESC lines would also be useful in the genetic
engineering of these animals for improved production
traits and products for disease resistance and biopharm-
ing. However, horses have the disadvantages of low
embryo numbers harvested during in vivo blastocysts
collection or in vitro embryo production (HacKett &
Fortier 2011). Saito et al. (2002) isolated for the first time
ES-like cells from equine blastocysts and induced their
in vitro differentiation. In human, different authors
observed that after ESCs in vivo implantation teratomas
have been developed. Li et al. (2006) injected equine
ES-like cells into several combined immune deficient
mice and observed no teratoma formation. However,
tumour formation by stem cells is more common
when they are injected into the same species from
which they were derived (Erdo et al. 2003). Recently,
the injection of undifferentiated equine ES-like cells
into experimentally induced tendon lesions has been
reported by Guest et al. (2010). In the study, at no time
was there any evidence of teratoma formation by the
horse ESCs, or differentiation into unwanted cell types;
however, longer follow-up periods in larger numbers
of horses are required to demonstrate full safety.
Accordingly, many scientists have focused on alterna-
tive sources of stem cells from various mesenchymal
tissues, such as bone marrow mesenchymal stem cells
(BMMSCs; Horwitz et al. 1999), adipose tissue (Seo
et al. 2005) and umbilical cord (UC; Romanov et al.
2003). MSCs have distinct cell–surface antigen markers.
q2012 Society for Reproduction and Fertility DOI: 10.1530/REP-10-0408
ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org
As reported by the International Society of Cellular
Therapy, MSC populations express CD73, CD90 and
CD105. CD73 is a purine catabolic enzyme with broad
substrate specificity that catalyses the dephosphorylation
of purine and pyrimidine ribo- and deoxyribonucleoside
monophosphates to their corresponding nucleosides
(Naito & Lowenstein 1981); furthermore, CD73 has
been postulated to play a role in cell adhesion (Airas
et al. 1995). CD90, also called Thy-1, is originally
discovered as a thymocyte antigen. Thy-1 can be used as
a marker for a variety of stem cells and for the axonal
processes of mature neurons, even if its function has not
yet been fully elucidated: it has speculated its roles in
cell–cell and cell–matrix interactions, with implication
in neurite outgrowth, nerve regeneration, apoptosis,
metastasis, inflammation and fibrosis (Nakamura et al.
2006). CD105, or endoglobin, is a well-known MSC
marker: it is a part of TGFbreceptor complex. It thus may
be involved in the binding of TGFb1, 3, etc. but it is also
involved in cytoskeletal organization affecting cell
morphology and migration (Dominici et al. 2006).
Additionally, MSCs lack expression of haematopoietic
markers, such as CD14, CD34 and CD45. CD45 is a
pan-leukocyte marker; CD34 markers are primitive
haematopoietic progenitors and endothelial cells; and
CD14 is prominently expressed in monocytes and
macrophages (Dominici et al. 2006).
The difficulties with bone marrow are obtaining tissue
samples from donors and finding a significant decrease
in both quality and differentiation potential of BMMSCs
with age donor. Widely disparate results have been
reported with regard to MSC numbers, differentiation
potency and aging. A negative correlation between
donor age, number and proliferative capacity of MSCs
isolated from young and old donors has been demon-
strated by several authors (Martin et al. 1970,Schneider
& Mitsui 1976,Majors et al. 1997,D’Ippolito et al.
1999,Stenderup et al. 2003,Mareschi et al. 2006), but
widely different results have been obtained with regard
to MSC antigen expression. Recently, Stolzing et al.
(2008) confirmed those already demonstrated in
previous studies (D’Ippolito et al. 1999,Mendes et al.
2002,Chen et al. 2005,Zhang et al. 2005,Zheng
et al. 2007,Kretlow et al. 2008), observing a significant
age-related change in membrane markers expression
levels, an alkaline phosphatase activity, chondrogenic
and myogenic differentiation potency decline with
donor age in contrast with adipogenic differentiation.
However, these data are in contrast with those reported
by other research groups (Bergman et al.1996,
Stenderup et al. 2001,2003,Bellows et al. 2003),
which showed no age-related differences in differen-
tiation potency. Different results obtained are with regard
to the choice of age groups, group size, gender, inclusion
of pathological states, isolation and cultivation con-
ditions (Sethe et al. 2006). In equine medicine, only
Colleoni et al. (2009) reported a significant decline in the
yield of cells from samples of two old horses (8 and 9
years old); however, due to the low number of samples
examined in this study, data need to be further
confirmed.
Beyond natural sources that are limited by stem cell
availability, immune intolerance and lineage specifi-
cation, the latest platform for recently developed
bioengineered stem cells are IPS cells (Nelson et al.
2010). IPS cells generation has been reported for mouse
(Takahashi & Yamanaka 2006), human (Park et al. 2008),
rat (Liao et al. 2009), monkey (Liu et al. 2008)and
recently in the horse (Nagy et al. 2011), suggesting that
virtually any mammalian species can be used for IPS cell
derivation. With the advent of IPS technology, the
limitations related to the use of ESCs and MSCs could
be addressed by the pluripotent potential of bioengi-
neered stem cells that are derived from autologous
sources. Utilizing IPS-based technology, all lineages of
the adult body have become viable targets for replace-
ment. IPS cells enable the ability to genetically repair
sequence defects through homologous recombination,
which then produces healthy stem cells devoid of the
original disease causing genetic impairment. Thereby,
the ability to reproducibly generate unlimited self-
derived progenitors that avoid immune intolerance is a
unique feature. However, the unlimited differentiation
potential of IPS is similar to ESCs, and thus the risk of
dysregulated growth and teratoma formation requires
stringent safeguards (Nelson et al. 2008). Moreover,
beyond the common challenges of natural pluripotent
stem cells, IPS cells also contain genetic modification as
a consequence of the strategy used for reprogramming or
spontaneously acquired cytogenetic abnormalities due
to extensive in vitro manipulation. The long-term
implications of nuclear reprogramming are yet to be
determined as this technology is in the early stages of
development.
The disadvantages of ESCs, BMMSCs and IPS have
accelerated the search for alternative sources of stem
cells as well as UC blood (UCB), Wharton’s jelly (WJ)
and amniotic fluid (AF). In human medicine, the use of
UCB tissue as a source of MSCs can be traced back to
2000: in this year Erices et al. (2000) showed that cells
from UCB were able to give rise to two types of adherent
cells, one of which expressed antigens typical of MSCs.
Equine UCBMSCs have been isolated for the first time by
Koch et al. (2007). Authors observed that isolated cells
were able to differentiate in vitro into osteocyte,
adipocyte and chondrocyte (Koch et al. 2007), but no
studies on their molecular characterization have been
performed. Only recently, Lovati et al. (2011), using
RT-PCR, observed that MSCs from UCB expressed
CD105, CD29 and CD44, and were negative for
haematopoietic marker CD34. No further investigations
have been carried out on equine UCBMSCs molecular
characterization.
456 E Iacono and others
Reproduction (2012) 143 455–468 www.reproduction-online.org
The UC intervascular stroma consists of so-called
‘mucous’ or mesenchymal connective tissue, also
called Wharton’s Jelly (WJ). WJ develops from extra-
embryonic mesoderm, binds and encases the umbilical
vessels, protecting them from twisting and compression
during pregnancy. It also has angiogenic and metabolic
roles for the umbilical circulation. Since 1990, stromal
cells, which basically resemble mesenchymal fibroblasts
found elsewhere during in utero development, were
identified in human WJ (Takechi et al. 1993,Nanaev
et al.1997). Stemness of these cells has been
demonstrated by in vitro differentiation (Lu et al. 2006)
and identification of MSCs membrane-specific markers.
Furthermore, these cells did not express haematopoietic
lineage markers such as CD34, CD45 and CD14
(Wang et al. 2004,Weiss et al. 2006,Secco et al.
2008). In the equine species, WJMSCs have been
isolated in 2007 by Hoynowski et al. Authors identified
isolated cells like MSCs due to their osteogenic,
chondrogenic and adipogenic potency when cultured
in induction media. As suggested by Dominici et al.
(2006), the expression of markers associated with stem
cells from embryonic and ASCs has been observed by
flow cytometry (Hoynowski et al. 2007).
Since 1980, multiple cell types from embryonic and
extra-embryonic tissues were identified in AF (Gosden
1983), but only in 2000 cells with an immunocyto-
chemical profile of mesenchymal, fibroblast/myofibro-
blast cell lineages have been isolated and expanded in
vitro (Kaviani et al. 2001). Prusa et al. (2004) observed
the presence of a cell distinct population expressing
POU5F1 (Oct4), a marker for pluripotent stem cells
known to be expressed in ESCs, in human AF (Pesce &
Scholer 2001). The mechanisms involved in the POU5F1
regulation in ESC have been widely investigated, but in
the adult stage (including post-natal and fetus stages)
POU5F1 function has not been identified even though it
has been associated with the undifferentiated pluripotent
state of stem cell populations derived from various adult
human tissues or organs such as bone marrow-derived
multipotent adult progenitor cells. Furthermore, positive
expression of mesenchymal markers such as CD90,
CD105 and CD73; and negative expression of haema-
topoietic markers such as CD45, CD34 and CD14 have
been observed (In’t Aker et al. 2003,Tsai et al. 2004,De
Coppi et al. 2007). Recently, De Coppi et al. (2007)
showed that human and rodent AF-derived stem cells
(AFMSCs) are positive for c-kit (CD117), that is also
expressed in the heart (Beltrami et al. 2003) and retina
stem cells (Koso et al. 2007), indicating that the c-kit
receptor can identify a stem cell population within
different organs. The c-kit receptor is a protein tyrosine
kinase that binds a cytokine named stem cell factor.
This cytokine and its receptor have been involved
in embryogenesis, carcinogenesis, spermatogenesis,
melanogenesis and play an important role in the
haematopoiesis during embryo development. AFMSCs
are thought to originate from the developing fetus and be
in an intermediate stage between ESCs and lineage-
restricted ASCs (De Coppi et al. 2007), but the exact
origin of these cells is unclear. However, due to their
adipogenic, osteogenic, myogenic, endothelial, neuro-
genic, hepatic, chondrogenic and renal differentiation
potency (Tsai et al. 2004,Perin et al. 2007), AFMSCs
may be a readily available source similar to ESCs for
large numbers of different cells progenitors (De Coppi
et al. 2007). In addition, AFMSCs have not shown
tumorigenicity, thus making them an exciting new
source of regenerative cells (Yo u et al. 2008).
Although there are promising results in human field,
few studies are available on characterization and
differentiation potential of these cells in domestic
animals. The aim of this study was to optimize the
isolation and culture of equine MSCs from AF, UCB and
WJ. Self-renewal ability and multilineage differentiation
capability have been further evaluated. We herein aimed
to widen the knowledge of equine MSCs from fetal fluid
and membranes characterization by testing several
molecular markers by flow cytometry. The markers
tested in the present study were chosen among those
suggested by the International Society for Cytotherapy
(ISC; Dominici et al. 2006). In addition, CD44 was
screened because of its role in MSC migration (Sackstein
et al. 2008) and its correlation with ‘stemness’ in other
MSC populations (Strem et al. 2005). Due to no
commercially available species-specific antibodies exist-
ence to characterize equine stem cells, in the present
study antibodies for human markers were involved using
cross-reactivity of the antibodies among different
species, such as recently reported by Park et al. (2011).
By an increasingly more detailed analysis of the equine
AFMSC phenotypic profile we hope that, as has been
done for human MSCs, a list of minimum cultural and
phenotypic criteria can be drawn up as soon as possible.
Results
Animals
In the equine species, the fetus was normally born in
anterior presentation, dorsal position and extended
(head, neck and forelimbs) posture. Failure to observe
the fluid-filled amnion (which may be visible only during
contractions) protruding from the vulva after 5 min from
the beginning of the second stage of parturition indicates
that we are probably facing a dystocia. In the present
work, AF, UCB and WJ have been recovered from mares
that underwent an eutocic (normal) parturition, without
induction, and which delivered healthy, viable foals, as
showed by an APGAR score R9(Vaala 2006). Samples
were obtained from 13 mares and no complications for
both mares and foals were encountered upon AF, CB and
UC sampling at delivery.
MSCs from equine amniotic fluid 457
www.reproduction-online.org Reproduction (2012) 143 455–468
Sampling and cell culture
AF samples from 10/13 (77.0%) donor mares have
been recovered: on average, we obtained 35.0
G23.5 ml/mare (range: 15–50 ml). We isolated cells
using a Percoll density-gradient centrifugation for elim-
inating urinary crystals, deposits of minerals and small
debris, which are present in AF during last gestation period
and can damage cells during in vitro culture. To obtain
the clear buffy coat layer, AF was diluted with PBS.
After washing pellets were seeded in culture dishes.
Adherent fibroblastoid spindle-shaped cells growing in
monolayer were isolated in 7/10 (70.0%) samples (Fig. 1A).
UCB samples (15–55 ml; mean 36.7G13.9 ml) were
recovered from 8/13 mares (61.5%), due to the early
umbilical vein collapse. From equine UCB, cells have
been isolated by Percoll density-gradient: to obtain a
clear buffy coat, UCB was diluted with PBS and
harvested mononuclear cells were washed several
times to remove platelets and red blood cells. Adherent
spindle-shaped cells have been isolated in 6/8 (75.0%)
samples (Fig. 1B); and in one sample (1/8: 12.5%) no
adherent cells were observed.
Immediately after foal detachment, UC samples have
been recovered (length w15 cm), after 13/13 (100%)
deliveries. UC samples were rinsed in antibiotic and
ethanol solutions and WJ has been isolated: the mean
weight of recovered jelly was 5.0G3.7 g (range: 1.8–
11 g). The jelly was minced and digested using an
enzymatic solution; after washing in PBS plus fetal
bovine serum (FBS), mononuclear cells were seeded in
culture dishes. Adherent cells with MSC morphology
have been isolated in 8/12 (66.7%) samples (Fig. 1C);
and 1/13 (7.7%) of collected UC was not processed for
cell isolation due to the absence of WJ.
The partum canal and delivery environment rep-
resent a non-sterile condition for horses. As a
consequence UC and amniotic tissue are exposed to
significant bacterial and yeasts contaminations. To
prevent from these contaminations, for AF and UCB
sampling, we used sterilized syringes and gloves and
pulled down AF quickly. Despite the precautions used,
in 3/10 (30.0%) AF samples no adherent cells have
been observed due to bacteria (1/3) and yeasts (2/3)
contaminations. On the contrary, only in 1/8 (12.5%)
UCB samples no adherent cells have been observed.
Lower isolation rate due to external contaminations has
been observed in WJ (4/12 samples: 33.3%). In this
study, to avoid contaminations without killing cells,
UC samples were immersed in a 70% ethanol solution
for 10 min but it was not sufficient to completely
eliminate contaminations.
Undifferentiated cells of all lines have been passaged
up to eight times and population-doubling times (DTs)
have been calculated. DT assay showed that AF, UCB
and WJMSCs were able to divide for an extensive period
in vitro. During P0 to P8, AF cells showed a mean DT of
2.3G1.0 days/CD (range: 0.6–4.5 days; Fig. 2A). By P8,
total mean CD was 37.3G3.0 (Fig. 2B). One line of
equine AFMSCs was cultured up to 118 days (15
passages) during which the cells reached 58.7 CD.
Considering the same culture passages (P0–P8), the
mean DT showed by equine UCBMSCs was 2.6
G1.3 days/CD (range: 1.1–5 days; Fig. 2C) and it was
statistically higher (P!0.05) than that showed by equine
AFMSCs (2.3G1.0 days/CD). By P8, UCBMSCs cell-
doubling number (CD) was 34.4G2.3 (Fig. 2D). Adher-
ent spindle-shaped cells isolated from equine WJ
showed a mean DT (2.0G0.6; range: 1.2–4 days;
Fig. 2E) significantly lower than those observed in
A
B
C
Figure 1 Monolayer of rapidly expanding adherent spindle-shaped
fibroblastoid cells compatible with undifferentiated MSC. (A) AF,
(B) UCB and (C) WJ. Magnification !10.
458 E Iacono and others
Reproduction (2012) 143 455–468 www.reproduction-online.org
AFMSCs and UCBMSCs culture (2.0G0.6 vs 2.6
G1.3 days/CD vs 2.3G1.0 days/CD respectively;
P!0.05). In this cell line, by P8, CD was 37.4G2.0
(Fig. 2F). Despite different DTs, no statistically significant
differences have been found in CD among the three
different cell lines. Furthermore, no lag phase has been
observed during in vitro culture of AF, UCB and
WJMSCs: in fact no statistically significant differences
in the number of CD have been found among different
culture passages (PO0.05). The results obtained indicate
that equine AFMSCs, UCBMSCs and WJMSCs had self-
renewal capacity.
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
01234
Passage
CD time (days/CD)
5678
45
40
35
30
25
20
15
10
5
0
01234
Passage
Total CD number
5678
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
01234
Passage
CD time (days/CD)
5678
40
35
30
25
20
15
10
5
0
01234
Passage
Total CD number
5678
01234
Passa
g
e
CD time (days/CD)
Cell doubling time equine
wharton's jelly MSCs (
n
=6)
Total cell doubling number equine
wharton's jelly MSCs (
n
=6)
Cell doubling time equine
wharton's jelly MSCs (
n
=4)
Cell doubling time amniotic
fluid MSCs (
n
=4)
AB
CD
EF
Total cell doubling number equine
equine cord blood MSCs (
n
=4)
Total cell doubling number equine
amniotic fluid MSCs (
n
=4)
5678
45
40
35
30
25
20
15
10
5
0
01234
Passa
g
e
Total CD number
5678
Figure 2 CT and CD of cultured primary and passaged MSCs. All values reflect the meanGS.D. (A and B) AFMSCs: data were compiled from seven
cell cultures. DT of primary cells and subsequent passages was not significantly different (PO0.05). The mean MSC doubling number at P8 was
37.3G3.0. (C and D) UCBMSCs: data were obtained from four cell cultures. DT of primary cells and subsequent passages was not significantly
different (PO0.05). The mean MSC doubling number at P8 was 34.4G2.3. (E and F) WJMSCs: data were compiled from six cell cultures. DT of
primary cells and subsequent passages was not significantly different (PO0.05). The mean MSC doubling number at P8 was 37.4G2.0.
MSCs from equine amniotic fluid 459
www.reproduction-online.org Reproduction (2012) 143 455–468
Immunophenotypic characterization by flow cytometry
ISC postulated that MSCs show positive expression for
CD73, CD90 and CD105; and negative expression for
CD14, CD34 and CD45 (Dominici et al. 2006). As there
are no equine-specific antibodies for flow cytometry, in
the present study we used anti-human antibodies,
routinely used by Immunohaematology and Transfusion
Center equipe, using cross-reactivity of antibodies
among different species. The antibodies efficiency was
verified by performing a control on circulating equine
lymphocytes. As expected, considering the results
obtained with human lymphocytes, adult and haemato-
poietic markers used have not been expressed by these
cells. Unexpected data have been registered for CD45
and CD73 that were negative also for lymphocytes (data
not shown). Furthermore, we compared amino acid
sequences using Basic Local Alignment Search Tool
(Blast). Our results, as summarized in Ta b le 1, showed a
similar expression pattern of MSC phenotype, but
different levels of reactivity of antibodies tested have
been observed. In particular, cells were reactive to
surface markers CD90 and CD105 in all cell lines
evaluated (Fig. 3). MSCs also demonstrated a marker
reaction with CD44 (Table 1 and Fig. 3), a cell–surface
glycoprotein having a role in MSCs migration. Others
markers for haematopoietic cell were not expressed
(Table 1 and Fig. 3). Due to negative lymphocytes CD45
and CD73 expression and the lack of horse CD45 and
CD73 sequence, for these markers cross-reactivity could
not be confirmed, as well as its negative expression by
equine MSCs.
In vitro differentiation
To characterize isolated cells, we further performed
trilineage differentiation assay. According to ISC, we
induced chondrogenic, osteogenic and adipogenic
differentiation culturing each lineage for several days in
induction media.
Chondrogenic induction has been performed in
monolayer culture and after 21 days cell Alcian Blue
staining has been performed to assess differentiation.
Alcian Blue staining is usually used to confirm
mucosubstances and acetic mucins deposition. We
found that induced cells were positive for Alcian Blue
(Fig. 4A). The negative controls kept in regular culture
medium showed no change in their morphology, no cells
stained positive for Alcian Blue (Fig. 4B).
Table 1 Flow cytometry analysis of P3 MSCs from AF, UCB and WJ for the surface markers CD90, CD105, CD73, CD44, CD14, CD34 and CD45.
Tissue CD90 (%) CD105 (%) CD73 (%) CD44 (%) CD14 (%) CD34 (%) CD45 (%)
AF 82.2 78.1 4.0 98.4 0.9 1.8 4.2
UCB 90.3 83.2 0.1 96.9 0.7 7.5 7.9
WJ 93.4 93.2 0.0 97.2 0.2 0.4 0.4
Isotype PE
CD105 CD44
AF CB WJ
CD90
CD90
CD90
CD105
CD105 CD44
CD44
CD34
CD45
CD73 CD73
CD45
CD34
CD73
CD34
CD45
Isotype APC Isotype
Figure 3 Overlay histograms of cytometry
analysis. In black isotypic controls are
represented. Empty histograms represent
the analysis with mAbs on mesenchymal
cell culture.
460 E Iacono and others
Reproduction (2012) 143 455–468 www.reproduction-online.org
To determine osteogenesis, isolated cells were
cultured in osteogenic induction medium and growth
medium was used as a control. Cells were maintained
in induction condition for 3 weeks. Significant calcium
depositions were detected in induced cells compared to
control using Von Kossa staining and Alizarin Red
(Fig. 4C and D).
To show adipogenic potency, cells were cultured
in adipogenic induction medium for 3 weeks. To detect
fat droplets and quantify adipogenesis, we stained cells
with Oil Red O, a fat-soluble dye. Cells maintained
in induction medium resulted positive for Oil Red O, while
undifferentiated control cells did not stain for fat droplets.
UCBMSCs showed a greater adipogenic potential than
AFMSCs and WJMSCs, characterized by a greater
accumulation of lipid vacuoles. Further investigation
using RT-PCR is needed to verify if different differentiation
capacities exist among these three cell lines.
AB
C
EF
GH
D
Figure 4 In vitro differentiation studies.
(A) Chondrogenic induction in equine AF
progenitors over 3 weeks: Alcian Blue
staining of glycosaminoglycans in cartilage
matrix. Magnification !10. (B) Chondro-
genic control: equine AF cells cultured in
regular medium for 21 days maintained
normal morphology and stained negative
for Alcian Blue. Magnification !10.
(C) Osteogenic induction in equine WJ
progenitors over 3 weeks: von Kossa
staining of extensive extracellular calcium
deposition. Magnification !20.
(D) Osteogenic control: equine WJ cells
cultured in standard medium for 21 days
maintained normal morphology and
stained negative for von Kossa staining.
Magnification !10. (E) Osteogenic induction
in equine AF progenitors over 3 weeks:
Alizarin Red S staining of extensive extra-
cellular calcium deposition. Magnification
!10. (F) Osteogenic control: equine AF cells
cultured in regular medium, after 21 days
presented normal morphology and stained
negative for Alizarin Red S staining.
Magnification !10. (G) Adipogenic induction
in equine CB progenitors over 3 weeks: Oil red
O staining of extensive intra-cellular lipid
droplet accumulation. Magnification !20.
(H) Adipogenic control: equine CB cells,
after 21 days of culture in standard
medium presented normal morphology and
stained negative for Oil red O staining.
Magnification !10.
MSCs from equine amniotic fluid 461
www.reproduction-online.org Reproduction (2012) 143 455–468
Discussion
Isolation and characterization of stem cells derived from
various tissues and sources are important issues in stem
cell field. WJ is the mature connective tissue surrounding
umbilical vessels: it is composed of myofibroblast-like
stromal cells, collagen fibres and proteoglycans
(Kobayashi et al. 1998). Recently, researchers have
focused on it as a potential new source of MSCs both
in humans and horses (Hoynowski et al. 2007,
Karahuseyinoglu et al. 2007,Passeri et al. 2009), but
the efficient isolation of cells that truly express MSC
characteristics has been somewhat controversial. Con-
troversial results have also been obtained for cells
isolated from equine UCB (Kock et al. 2007, Cremonesi
et al. 2008), and no report has been found on their
characterization using criteria postulated by ISC in this
species. Due to its contact with the developing fetus, AF
contains large numbers of suspended cells including
stem cells (In’t Anker et al. 2003,De Coppi et al. 2007).
To our knowledge, recently, only two studies have been
published on horse AF-derived progenitor cells, whereas
it is considered a rich source of MSCs in human beings.
We compared WJ, AF and UCB as viable, accessible and
not risky sources of MSCs in equine. In the present study
high initial sample volumes of AF, UCB and tissue have
been easily collected at the delivery, without any
invasiveness for the mare or for the foal, and high
numbers of viable nucleated cells from these sources
have been isolated, except for some failure samples
occurring in yeasts/bacterial contaminations as pre-
viously reported by other authors (Passeri et al. 2009,
Lovati et al. 2011). The isolation rate obtained in the
present study by centrifuging UCB on a Percoll density-
gradient was lower than that obtained by Koch et al.
(2009) using a PrepaCyte-EQ density gradient (100%).
Given the influence that laboratory conditions may
have on the outcome of projects, in the future it would
be appropriate to compare, under the same conditions,
the isolation method used in this study with those
reported by other authors. On the other hand, data
reported for human UCB (Kern et al. 2006) are lower
than that of ours. This difference could be due to
various factors, such as duration of parturition,
recovery protocol, time between sample collection
and processing in the laboratory: these factors
determined viable cells reduction because of their
transition to the fetus or their death.
We cultured cells isolated from equine AF, WJ and
UCB for several days, from P0 to P8, and they showed
a typical MSC morphology, having spindle shape
and adherent characteristics, as postulated by ISC
(Dominici et al. 2006). Regarding cells proliferation,
WJMSCs showed a higher expansion rate compared to
UCBMSCs and AFMSCs: results observed are similar
to those reported in human medicine for UCBMSCs
and WJMSCs (Sarugaser et al. 2005,Karahuseyinoglu
et al. 2007), but not for AFMSCs (Tsai et al. 2004).
Different DTs between human and horse AFMSCs
could be determined during the period of pregnancy in
which the drawing was made. Human AF samples
were recovered by amniocentesis, at the beginning of
the second trimester of pregnancy, therefore, human
AFMSCs could show DT more similar to ESCs.
UCBMSCs DT registered in our laboratory was higher
than that reported by Koch Research Group (Koch
et al. 2007,2009). The difference could be determined
by the higher concentration of FBS used by the authors
during in vitro culture (10 vs 30%). FBS is a mixture
of high-molecular-weight molecules that may promote
or inhibit cell growth and differentiation (Barnes et al.
1987,van der Valk et al. 2004); however, this
composition is largely unknown in both qualitative
and quantitative terms, and moreover FBS may be a
vehicle of known pathogens and unknown infectious
agents (Schallmoser et al. 2007). Different from that
observed by Lovati et al. (2011) and Eloit (1999),inour
study all cell lineages showed a similar fold expansion
during all passages (P0–P8) and WJMSCs were not
affected by the trypsin detachment as reported by the
other authors. The discrepancy in WJMSCs expansion
is likely due not only to different culture conditions,
but also to the different selections of cord tissue to be
processed. The immature cells that retain the ability to
proliferate were located close to the amniotic surface,
whereas highly differentiated, non-proliferating fibro-
blasts were located in closer proximity to the umbilical
vessels (Nanaev et al. 1997): in our study, only cord
jelly, closer to amniotic surface, was digested, while
Lovati et al. (2011) did not distinguish between near or
far from umbilical vessel portion thus the cell
population cultured was very heterogeneous and this
could explain the lower cell doubling.
In the present study, we also demonstrated the
multipotent capability of equine AFMSCs, UCBMSCs
and WJMSCs. The choice of lineages differentiation
was made based on the principles established by the
ISC (Dominici et al.2006). Chondrogenic and
osteogenic differentiations were carried out with a
view to future clinical application of these cells in
injured horses, as previously supported by Berg et al.
(2009). All three cell lines differentiated in chondro-
blasts, osteoblasts and adipocytes, as demonstrated by
staining, performed after 21 days of culture in
appropriate medium; control cells did not stain at all
and only the background colour remained. While a
lack, also testing different protocol, in adipogenic
efficiency of equine AFMSCs has been reported in a
previous study (Park et al. 2011), in our experiment
equine AFMSCs showed an adipogenic potential
similar to that observed in equine WJMSCs, while
UCBMSCs seem to show a greater adipogenic potential
than AFMSCs and WJMSCs, characterized by a
greater accumulation of lipid vacuoles. Further studies,
462 E Iacono and others
Reproduction (2012) 143 455–468 www.reproduction-online.org
using different induction protocols and evaluating
differentiation assay by cytology and RT-PCR, are
necessary to verify if these different findings are due
to different laboratory conditions or if in equine the
conditions for AF, WJ and UCBMSCs promote one type
of differentiation lineage rather than other, as suggested
by Park et al. (2011).
In the present work, phenotypic characterization of
equine AF, UCB and WJMSCs was carried out by the
expression analysis of several surface MSC markers. We
choose to investigate cells at the third culture passage,
as they have reached a sufficient level of homogeneity
at this stage. As there are no equine-specific antibodies
for flow cytometry, we used antibodies for human
markers. To provide evidence for inter-species cross-
reactivity, the similarity of CD markers was identified
comparing the amino acid sequence, as already been
reported by de Mattos Carvalho et al. (2009).
Furthermore, as suggested recently by Pascucci et al.
(2011) we used equine circulating lymphocytes as
control. In the present study, high percentage of equine
AF, UCB and WJ cells population expressed MSC
surface markers CD90 and CD105. In addition, flow
cytometry revealed that the majority of analysed cells
were positive also for CD44. Our data are similar to
those reported by Park et al. (2011) for equine AFMSCs
tested using anti-human antibodies; moreover, these
markers are present in human MSCs and have also
been found in equine MSCs isolated from adipose
tissue (de Mattos Carvalho et al. 2009,Pascucci et al.
2011). These findings confirm that concerning CD90
and CD44 a high phenotypic similarity do exist among
MSCs isolated from different species and sources (Strem
et al. 2005).
A lack of reactivity with haematopoietic markers
CD34 and CD14, which cross-reaction was confirmed
by lymphocytes investigation, indicates that isolated
cells are negative for haematopoietic progenitors. On the
other hand, the lack of reactivity of equine cells and
lymphocytes with the haematopoietic markers CD45
and MSC with marker CD73 probably indicates that the
human-directed reagents do not cross-react with their
corresponding equine epitopes. These findings need
further investigation to assess if, in particularly, the lack
of CD73 expression is due to the lack of cross-reactivity
or is a species-specific feature as interpreted by Pascucci
et al. (2011).
From results obtained in this study, we suggest the
existence of alternative sources of multipotent cells in
equine. Differentiation of AF, UCB and WJMSCs into
adipocytes and, above all, into osteocytes and chon-
drocytes underlies their possible use for regenerative
medicine in injured horses. Further study will be
necessary to evaluate the expression of cell antigens
and the possible use of these tissues as allogenic source
of MSCs.
Materials and Methods
Materials
All chemicals were obtained from Sigma–Aldrich, plastic
dishes and tubes from Sarstedt, Inc. (Newton, NC, USA) unless
otherwise noted.
Animals
Samples were obtained from 13 standardbred mares, between
6 and 15 years of age, belonging to one stable location near
the Faculty of Veterinary Medicine of Bologna University, or
housed at the Equine Perinatology Unit ‘Stefano Belluzzi’,
University of Bologna, for attending delivery. Experimental
procedures were carried out in accordance with DL 116/92
and were approved by the Ethics Committee at the University
of Bologna and by the Ministry of Health.
Sampling and MSCs isolation
Amniotic fluid
AF samples were taken soon after foal or placenta membranes
passed through vulva, using a sterile 18 gauge needle mounted
on a 60 ml sterile syringe (IMI, Padua, Italy), containing 1 ml
of EDTA solution to prevent clot formation that could affect
cell isolation. Samples were stored at 4 8Candfurther
processed within 12 h. Briefly, each sample was diluted 1:1
with Dulbecco’s Phosphate Buffer Solution (DPBS) containing
100 IU/ml penicillin and 100 mg/ml streptomycin. The
obtained solution was centrifuged (Heraeus Megafuge 1.0R;
rotor: Heraeus #2704; ThermoFisher Scientific Inc.) for 15 min
at 470 g. Supernatant was carefully removed and the pellet was
re-suspended in 5 ml of culture medium containing DMEM and
TCM199 (1:1), 10% (v/v) FBS (Gibco; Invitrogen Corporation),
100 IU/ml penicillin and 100 mg/ml streptomycin. Cells were
isolated by carefully loading sample on 5 ml of 70% Percoll
solution in a 50 ml polypropylene tube, centrifuging for 30 min
at 25 8C at 1880 g; the interphase was collected after aspirating
and discarding the supernatant, washed with 20 ml of culture
medium and centrifuged at 470 gfor 10 min at 25 8C. The
supernatant was aspirated and cells were washed with culture
medium a second and third time. Cells were then re-suspended
in 1 ml of culture medium and counted by haemocytometer.
Umbilical cord blood
CB was collected immediately after foaling and before the
UC breaks spontaneously or was broken according to
management protocol. Venipuncture of the umbilical vein
was performed with a 21 guage hypodermic needle attached
to a 60 ml sterile syringe (IMI), containing 1 ml of heparin
(Eparina Vister 5000 IU/ml; Marvecs Pharma, Milan, Italy) as
anti-coagulant. Blood samples were stored at 4 8C for at the
least 12 h. Each sample was diluted 1:1 with DPBS
containing 100 IU/ml penicillin and 100 mg/ml streptomycin.
The solution was centrifuged (Heraeus Megafuge 1.0R; rotor:
Heraeus #2704) for 15 min at 470 g. Supernatant was
removed and the pellet was re-suspended in 5 ml of culture
medium. The mononuclear cell fraction was isolated by
MSCs from equine amniotic fluid 463
www.reproduction-online.org Reproduction (2012) 143 455–468
carefully loading sample on 5 ml of 70% Percoll solution in a
50 ml tube, centrifuging for 30 min at 1880 gat 25 8C. The
interphase was then collected after aspirating and discarding
the supernatant washed with 20 ml of culture medium by
centrifuging at 470 gfor 10 min at 25 8C. The supernatant
was aspirated and cells were washed twice with culture
medium. Cells were then re-suspended in 1 ml of culture
medium and counted by haemocytometer.
Wharton’s Jelly
Immediately after breaking the UC, the part closest to the
Colt, characterized by an abundant amount of WJ, was
severed. Samples were stored in DPBS containing 100 IU/ml
penicillin and 100 mg/ml streptomycin, at 4 8C for at the latest
12 h. UC was disinfected by immersing for 10 min in 70%
ethanol. In a laminar flow hood, tissue was rinsed by
repeated immersion in DPBS and WJ was isolated, weighed
and minced finely (0.5 cm) by sterile scissors. Minced WJ
was transferred into a 50 ml polypropylene tube, to which
was added 1 ml/1 g sample of a digestion solution (0.1%
(w/v) collagenase type I (Gibco, Invitrogen Corporation),
dissolved in DPBS solution and sterilely filtered). The tissue
and digestion solution were mixed thoroughly, incubated in a
37 8C water bath for 1–2 h, and mixed every 15 min. After
incubation, collagenase was inactivated by diluting 1:1 with
DPBS plus 10% (v/v) FBS. The solution obtained was filtered
and undigested tissue was discarded. Nucleated cells were
pelleted at 470 gfor 10 min. The supernatant was discarded,
pellet was re-suspended in 5 ml of culture medium and spun
at 470 gfor 10 min to wash cells. This operation was
repeated three times. After the last wash, cell pellet was
re-suspended in 1 ml of culture medium and cell concen-
tration was counted by haemocytometer.
Cell doubling method
Primary cells were plated in a 25 cm
2
flask at a maximum
density of 5!10
4
cells/cm
2
and incubated in a 5% CO
2
humidified atmosphere at 38.5 8C. The medium was
completely replaced after 48 h in order to remove non-
adherent cells. Next the medium was completely replaced
every 3 days until the adherent cell population reached w80%
confluence. At this point, the adherent primary MSCs were
passaged by digestion with 0.05% (w/v) trypsin, counted
with a haemocytometer, and re-seeded as ‘Passage 1’ (P1) at
25!10
3
cells/cm
2
. For the subsequent passages, cells were
inoculated in 5 cm
2
flasks at 25!10
3
cells/cm
2
and allowed
to multiply for 6–7 days to 90% confluence before trypsini-
zation and successive passage. DT and CD were calculated
from haemocytometer counts and cell culture time (CT) for
each passage according to the following two formulae
(Vidal et al. 2007):
CD ZlnðNf=NiÞ=lnð2Þ(1)
DT ZCT=CD (2)
where N
f
is the final number of cells and N
i
the initial
number of cells.
Chondrogenic in vitro differentiation
Expanded cells were harvested after three passages, washed
in DPBS, and placed in a six-well plate at a density of 5!
10
3
cells/cm
2
in chondrogenic induction medium, consisting
of DMEM/TCM199, 100 IU/ml penicillin, 100 mg/ml strepto-
mycin, 6.25 mg/ml insulin, 50 nM ascorbate-2-phosphate,
0.1 mM dexamethasone, 10 ng/ml human transforming
growth factor (hTGF)-b1 and 1% (v/v) FBS. Cells in
monolayer were incubated for 3 weeks. As a negative
control an equal number of cells were cultured in expansion
media for 21 days. In both groups the medium was
completely replaced every 3 days. After 3 weeks of culture,
cells were fixed with 10% (v/v) formalin for 1 h at room
temperature (RT), then stained with Alcian Blue solution (1%
in 3% acetic acid (v/v), pH 2.5) for 15 min at RT. Alcian Blue
staining acid mucosubstances and acetic mucins confirmed
chondrogenic differentiation cytologically.
Osteogenic in vitro differentiation
Undifferentiated cells were induced towards the osteogenic
lineage using the protocol described by Mizuno & Hyaku-
soku (2003). Briefly, at passage 3 putative cells were seeded
in a six-well plate at a density of 5!10
3
cells/cm
2
and
cultured in base medium for 24 h to allow cell adhesion.
After cells had adhered to the plate, they were treated with
an osteogenic induction medium consisting of 10 mM
b-glycerophosphate, 0.1 mM dexamethasone, 50 mM ascor-
bate-2-phosphate and 10% (v/v) FBS in DMEM/TCM199.
DMEM/TCM199 supplemented with 10% FBS was used as a
control medium. Media in both groups were completely
changed twice a week. After 21 days of induction, cells were
stained to confirm osteogenic differentiation.
von Kossa staining
In the osteogenic assay, later stage of osteogenesis,
characterized by calcium deposition, was assessed 3 weeks
after induction via von Kossa and Alizarin Red S stainings
for detection of deposits of calcium or calcium salt.
For von Kossa staining, cells were washed three times
with PBS before cell fixation with 10% (v/v) formalin for
1 h at RT. Cells were washed five times with distilled water
before adding 1 ml of 5% (w/v) silver nitrate and exposing
to yellow light for 15 min. The wells were washed five times
with distilled water. Calcium phosphate deposits stained
black.
Alizarin Red S staining
To confirm osteogenic differentiation, Alizarin Red S staining
was also performed for detecting calcium deposition. In brief,
cells were rinsed with DPBS and fixed incubating in ice-cold
ethanol 70% (v/v) for 1 h at RT. After three washes with
distilled water 1 ml of 2% (w/v) Alizarin Red S (pH 4.1–4.3)
solution was added. The plate was incubated at RT for
30 min, then Alizarin Red S solution was removed and then
the cells were rinsed four times with distilled water.
464 E Iacono and others
Reproduction (2012) 143 455–468 www.reproduction-online.org
Adipogenic in vitro differentiation
Adipogenesis was induced using protocol described by Mizuno
& Hyakusoku (2003) and Koch et al. (2007). Six-well culture
plates were seeded at a density of 5!10
3
cells undifferentiated
MSCs per cm
2
of tissue culture surface area in 0.5 ml of
expansion medium per tissue culture surface. After 24 h,
medium was completely replaced with adipogenic induction
medium consisting of 15% (v/v) rabbit serum, 1 mM dexa-
methasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX),
10 mg/ml bovine insulin, 0.2 mM indomethacin in DMEM/
TCM199. After 72 h of culture, the IBMX supplement was
removed from the medium. After 3 additional days, the
dexamethasone supplement was removed from the medium,
and cells were cultured for additional 15 days. Equal cell
number was cultured in expansion medium for negative
control. Both groups were maintained in culture for a total of
21 days and media were completely exchanged every 3 days.
To asses adipogenic differentiation, cells were stained to
evaluate the baseline formation of neutral lipid vacuoles
stainable with Oil Red O. Medium was aspirated, cells were
washed once with PBS then fixed with 10% (v/v) formalin for
1 h at RT. The formalin was then replaced with 2 ml of sterile
water for a few minutes. Water was aspirated and replaced with
60% (v/v) isopropanol for 5 min and then cells were covered
with Oil Red O solution (0.3% in 60% isopropanol (v/v)). After
5 min, cells were rinsed with distilled water and lipid vacuoles
appeared red.
Characterization of MSCs
Cytofluorimetric analysis was performed to identify cell surface
marker expression of equine MSCs. At passage 3, cells were
labelled with the following mAbs: CD105, CD45, CD90,
CD44, CD34, CD14 and CD73 (all from Beckman Coulter,
Fullerton, CA, USA). Cells were also labelled with isotype
control antibodies. Briefly, at 80% confluence, cells at P3 were
washed twice with DPBS, harvested using 0.05% (w/v) trypsin
solution and aliquoted at a concentration of 0.5–10
6
cells/ml.
Each aliquot was fixed and permeabilized using Reagent 1 of
Intraprep Kit (Beckman Coulter, Miami, FL, USA) according to
the manufacturer’s instructions. Cells were stained for 30 min
with either conjugated-specific antibodies or istotype-matched
control mouse IgG (Table 2) at recommended concentrations.
Labelled cells were washed twice in DPBS and fluorescence
intensity was evaluated using an FC500 two-laser equipped
cytometer (Beckman Coulter). All analyses were based on
control cells incubated with isotype-specific IgGs to establish
the background signal. Cross-reactivity of the antibodies used
was screened using cultured human and horse MSCs.
Furthermore, to verify cross-reactivity, control of circulating
equine lymphocytes was carried out. The similarity of CD
markers was also identified by comparing the amino acid
sequences using Basic Local Alignment Search Tool (BLAST).
Results were further analysed with the CXP dedicated program.
Statistical analysis
CT and CD are expressed as meanGS.D. Statistical analysis was
performed using Statistics for Windows (Stat Soft, Inc., Tulsa,
OK, USA). Data were analysed using one-way ANOVA for
multiple comparisons. Significance has been assessed for
P!0.05.
Declaration of interest
The authors declare that there is no conflict of interest that
could be perceived as prejudicing the impartiality of the
reported research.
Funding
This research was supported by University of Bologna (RFO:
Ricerca Fondamentale Orientata).
Acknowledgements
The authors wish to thank Prof. Gaetano Mari (Department of
Veterinary Medical Sciences, University of Bologna, Ozzano
Emilia (BO), Italy), Dr Carolina Castagnetti (Equine Perinatol-
ogy Unit, Department of Veterinary Medical Sciences,
University of Bologna, Ozzano Emilia (BO) Italy) and Scuderia
Trio Srl (Ozzano Emilia (BO), Italy), for the agreement of the
sample collection. Thanks are also due to Dr Silvia Colleoni
(AVANTEA Srl, Cremona, Italy) and Prof. Cesare Galli
(Department of Veterinary Medical Scineces, University of
Bologna, Ozzano Emilia (BO), Italy) for their advice on cell
culture protocols.
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Received 28 September 2010
First decision 12 November 2010
Revised manuscript received 2 December 2011
Accepted 20 January 2012
468 E Iacono and others
Reproduction (2012) 143 455–468 www.reproduction-online.org
