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Review Article
Embryonic Stem Cells in Cattle
M Mun
˜oz
1
,CDı
´ez
1
, JN Caaman
˜o
1
, A Jouneau
2,3,4
, I Hue
2,3,4
and E Go
´mez
1
1
Area de Gene
´tica y Reproduccio
´n, SERIDA, Camino de los Claveles, Gijo
´n, Asturias, Spain;
2
INRA and
3
ENVA, UMR 1198;
4
CNRS, FRE 2857,
Biologie du Developpement et Reproduction, Jouy-en-Josas, France
Contents
Because of the potential use of embryonic stem cells (ESC),
especially for genetic modifications, there is great interest in
establishing domestic animals-related ESCs. Unfortunately,
despite considerable efforts, validated ESC lines in species
other than mice and primates are yet to be isolated. In this
paper, we will summarize the current knowledge on bovine
ESCs in an attempt to understand why derivation of domestic
animal ESC is still unsuccessful and we will discuss some
promising future approaches.
Introduction
Embryonic stem cells (ESCs) were first isolated from
mouse embryos over 20 years ago (Evans and Kaufman
1981; Martin 1981). These cells, which are able to self-
renew indefinitely and to differentiate in vitro and in vivo
into derivatives of all three germ layers, were initially
used to study the differentiation process. Yet, soon after,
it became clear that ESCs provided an efficient route for
precise modification of the genome by gene targeting.
The first mutant mice derived from genetically engi-
neered ESCs were created, opening the doors to a new
era of animal transgenesis (Capecchi 1989).
The successful isolation and multiple applications of
mouse ESC (mESC) resulted in numerous efforts aimed
to establish ESCs in other species. Livestock production,
for example, would greatly benefit if farm animals ESCs
were established, in order to efficiently create genetically
modified farm animals bearing improved production
traits or increased disease resistances. Furthermore,
ESC lines from farm animals such as pigs, sheep or
cattle will enable to create transgenic animals for
modelling human diseases without some of the limita-
tions from the mouse model, i.e. short life span or a
physiology and anatomy very different from humans.
Unfortunately, although considerable effort has been
exerted to isolate and maintain ESC lines from domestic
animals, validated ESC lines in species other that mice
and primates are yet to be established.
Issues that need to be investigated are many, including
the identification of species-specific mechanisms under-
lying pluripotency and markers (for review, see Keefer
et al. 2007). A further hurdle is that most of the current
empirical approaches to obtain ESC from farm animals
closely followed procedures that were developed in
humans and mice. Nevertheless, data extrapolation from
these species has produced unsatisfactory results and
might have misled researchers from finding the func-
tional pathways that control pluripotency in ungulates.
In this paper, we analyse some of the currently
available data on bovine ESCs (bESC) to have a better
understanding of the failure to establish domestic
animals-validated ESC lines. We also describe some
new approaches that will perhaps enable the establish-
ment of these cell lines.
The Stage and the Source of Embryos
Embryonic stem cell lines have been successfully
isolated from mouse, monkey and human blastocysts,
although outstanding derivations have also been made
using embryos at pre-compaction stages (Eistetter 1988;
Delhaise et al. 1996; Strelchenko 1996; Mitalipova et al.
2001). Most attempts to isolate and culture bESCs have
been done with day 7–9 bovine blastocysts (Stice et al.
1996; Strelchenko 1996; Cibelli et al. 1998; Iwasaki
et al. 2000; Betts et al. 2001; Saito et al. 2003; Roach
et al. 2006; Mun
˜oz et al. 2008) although ESC-like cells
have also being isolated from day 12–14 embryos
(Gjørret and Maddox-Hyttel 2005). Yet, the optimal
timing of bovine pre-implantation development to
derive ESCs is still unknown.
Attempts to derive bESC from zygotes and early
cleavage stage embryos mostly failed (Strelchenko 1996;
Mitalipova et al. 2001), while only a single bovine
embryonic cell line, generated from a two-cell embryo,
has been cultured over 3 years (Mitalipova et al. 2001).
Yet, when bovine morulae were used as starting
material, efficient colony formation rates ranged over
60–70% (Stice et al. 1996; Strelchenko 1996). The
former author found that the embryonic stage (morulae
and Day-7 blastocysts) or source (in vivo and in vitro)
used did not influence the efficiency in establishing bESC
colonies. These results are nevertheless contrary to
studies reporting that Day-8 hatched blastocysts yield
a higher proportion of epiblast colonies than inner cell
masses (ICMs) isolated from Day-9 blastocysts (41%
and 13%, respectively) (Talbot et al. 1995). Although
bovine embryos have been used at different pre-implan-
tation stages to isolate bESC, the lack of standardized
and detailed protocols leads to an absence of homoge-
neous criteria to define efficiency of colony formation.
This hurdle precludes comparisons between published
data, making it impossible to conclude which develop-
mental stage entails the best starting material to isolate
ESCs. An alternative approach to answer this question
might be to extrapolate available data on mouse
embryonic development and mESC derivation; yet
Reprod Dom Anim 43 (Suppl. 4), 32–37 (2008); doi: 10.1111/j.1439-0531.2008.01229.x
ISSN 0936-6768
2008 The Authors. Journal compilation 2008 Blackwell Verlag
several species-specific differences must be carefully
considered prior to doing so. In mouse embryos, the
first differentiation event occurs at the late morula stage,
when the outer cells adopt an epithelial structure. This
event is followed by the formation of the blastocoel,
which marks the divergence of the first two lineages:
trophectoderm (TE) and inner cell mass. Upon blasto-
cyst expansion, differentiation continues with the ICM
forming two further lineages: the epiblast and the
primitive endoderm or hypoblast. Although epiblast,
hypoblast and TE lineages will be present in blastocysts
of all mammalian embryos, the time between fertiliza-
tion and formation is shorter in mouse than in other
species. Therefore, in order to isolate ESC from the
same type of cells used to derive mESC, i.e. epiblast cells
(Brook and Gardner 1997), we must consider differences
in the time scale of development and make sure that
embryonic stages equivalent to the murine counterparts
are used. In the pig blastocyst a defined epiblast is not
present before hatching, while in mice the epiblast is
already formed in blastocysts enclosed in the zona
pellucida. Therefore, derivation of ESCs from early
murine or porcine blastocysts is not equivalent. In
addition to differences in time scale of development,
recent data have reported important differences in
pathways that control pluripotency vs extra-embryonic
lineage restriction in mouse (Rossant 2007) and bovine
(Degrelle et al. 2005) (see below characterization of
bESC). Therefore any data extrapolation between
mouse and late-implanting species such as cattle should
be carefully considered.
Bovine embryos from different sources have been used
to isolate bESCs. Yet the only published experiment
aimed to compare the feasibility of in vitro- and in vivo-
derived embryos for the isolation of pluripotent cells
was undertaken by Talbot in 1995. Such a study
demonstrated that in vivo-derived blastocysts, especially
from early hatching blastocysts, were shown to be a
source of pluripotent epiblasts superior to their in vitro-
produced (IVP) counterparts.
The basis for any advantage by in vivo-produced
blastocyst to produce ESC lines is not known,
although a number of differences in morphology,
metabolic rates, gene expression and susceptibility to
cooling damage (Smith et al. 2005; Lonergan et al.
2006) have been reported between in vivo-derived and
IVP bovine embryos. It is possible that the reduced
number of cells present in the ICM of IVP bovine
embryos (Van Soom et al. 1996) might affect survival
of the ICM in culture, hindering the chances to
establish ESC lines from IVP embryos. In fact,
Anderson et al. (1994) assumed that the factor that
may affect survival of porcine ICMs in culture was the
number of cells of the ICMs.
In vivo-derived embryos might be a better source of
pluripotent cells, but their use as a starting material to
isolate ESC is expensive and laborious. Therefore, it
would be advisable to improve the procedures to derive
ESCs from IVP embryos, as well as the ability of IVP-
ICMs to yield ESCs. An obvious way to progress in this
aspect would be increasing numbers of cells in these
ICMs.
Characterization of Bovine ESC
Morphology, as well as the capacity to differentiate
in vitro through embryoid body (EB) formation, was one
of the two defining criteria initially used to identify
bESC cultures. Other traits such as small size, rounded
shape or high nucleus to cytoplasm ratio were used to
define bESC lines. Yet, cells belonging to TE and
visceral endoderm, which usually can be found in
blastocysts or isolated ICMs primary cultures, may be
confounded with ESC if solely morphological features
are used as evaluating criteria. Bovine blastocyst-derived
TE and endoderm cell lines have been thoroughly
characterized not only by morphological criteria but
also by the expression tissue-specific marker. For
instance, transferrin is a definitive marker for bovine
blastocyst-derived endoderm cell lines (Talbot et al.
2000). Therefore, the combined use of morphological
criteria and the analysis of extra-embryonic markers is
suggested to trul y identify bESC and ⁄or rule out the
presence of TE or visceral endoderm cells in ESC
cultures.
A useful strategy to characterize ESC lines is to
analyze the expression of pluripotency-related molecular
markers. Unfortunately, until now, no specific markers
have been identified in bovine. Therefore, markers
associated to pluripotency in other species (heterospec-
ific pluripotency markers) such as stage-specific embry-
onic antigens (SSEA-1, -3, -4) have been used to
characterize bESC. SSEAs are developmentally regu-
lated cell surface antigens expressed by murine and
human pluripotent cells (Fig. 1). mESCs strongly
express SSEA-1 (Solter and Knowles 1978; Gooi et al.
SSEA4
TRA-1-81
Bovine blastocyst Human blastocyst Murine blastocyst
SSEA1
SSEA4
TRA-1-81
TRA-1-60
SSEA4
TRA-1-81
TRA-1-60 SSEA1 SSEA1
SSEA1
TRA-1-60
Bovine ESC Murine ESCHuman ESC
SSEA4
TRA-1-81
SSEA4
TRA-1-81
SSEA1
TRA-1-60
TRA 1 81
TRA-1-60
Fig. 1. Expression of stage specific
embryonic antigens SSEA-1 and
SSEA-4 and keratin sulphate-ass-
ociated antigens TRA-1-60 and
TRA-1-81 in human and murine
blastocyst and embryonic stem
cells and in bovine blastocysts and
bovine ESC-like cells. Data from
human and mouse ICM and trop-
hectoderm are from Henderson
et al. (2002); data from human and
mouse embryonic stem cells are
from Boiani and Scho
¨ler (1997).
Data from bovine are from our lab
ESCs in Cattle 33
2008 The Authors. Journal compilation 2008 Blackwell Verlag
1981), whereas differentiated mESCs are characterized
by the loss of SSEA-1 expression and in some instances,
by the appearance of SSEA-3 and SSEA-4 (Solter and
Knowles 1979). In contrast, hESCs typically express
SSEA-3 and SSEA-4, but not SSEA-1 and their differ-
entiation is characterized by down-regulation of SSEA-3
and SSEA-4 and up-regulation of SSEA-1 (Andrews
et al. 1984; Fenderson et al. 1987). Undifferentiated
hESCs also express the keratin sulphate-associated
antigens TRA-1-60 and TRA-1-81 (Andrews et al.
1984). In bovine, a positive staining for SSEA-1,
SSEA-3 and SSEA-4 was reported in three embryonic
cell lines derived from pre-compaction embryos (Mita-
lipova et al. 2001). Similarly, SSEA-1 expression was
also detected by Saito et al. (2003), while none of the
bovine ES-like cells analysed by these authors were
found positive for SSEA-3 or SSEA-4. In contrast,
Wang et al. (2005) reported a positive SSEA-4 staining
in the absence of SSEA-1 staining in five ESC lines.
We have reported positive staining for SSEA-4, TRA-
1-60 and TRA-1-81 in bESC-like cells (Mun
˜oz et al.
2008). Unfortunately, the above antigens were not only
present in the ICM of bovine blastocysts but also in the
TE (Fig. 1). Therefore in bovine, these markers are not
specific for undifferentiated and ⁄or pluripotent cells.
The use of such markers to characterize bESC may
mislead researchers into isolating and culturing TE-
derived cells instead of ESCs.
The expression of SSEAs, in the TE of bovine
blastocysts, was unexpected, considering that for a long
time SSEAs have been used to characterize ‘undifferen-
tiated’ bESCs. Nevertheless, it was not totally surprising
as bovine TE cells show a slow differentiating phenotype
characterized by the co-expression of epiblast-specifying
genes (OCT-4, SOX-2, NANOG) and proteins (OCT-4,
NANOG) and trophoblast-specific genes (CDX-2,
HAND1, ETS-2, IFN-TAU, C12) (Kirchhof et al. 2000;
Degrelle et al. 2005; Mun
˜oz et al. 2008). Therefore in
bovine, the expression of markers which are associated
to pluripotency in other species (SSEA-4, TRA-1-60,
TRA-1-81, OCT-4, NANOG) is not restricted to plurip-
otent cells (Figs 1 and 2), which is a warning to validate
any pluripotency marker before its heterospecific use.
An additional difficulty to characterize bESC is that
available antibodies currently used to characterize ESCs
are produced using mouse or human proteins as
immunogens. Therefore, their ability to cross-react with
the appropriated bovine protein should be evaluated
before their use.
Cell Culture Conditions
Culture conditions close to those established for murine
ESC culture were successfully used to derive monkey
(Thomson et al. 1995) and human ESCs (Thomson
et al. 1998). Nevertheless, it soon became evident that
some factors required for the maintenance of mESC
pluripotency were not only dispensable in maintaining
hESC pluripotency but were also detrimental. As an
example, this occurred with BMP4, a member of the
transforming growth factor-b(TGF-b) family involved
in controlling mESC differentiation that induces differ-
entiation of human ESCs into trophoblast cells (Xu
et al. 2002). Since then, considerable amount of data
have been published over differences between mouse and
human pluripotency maintaining factors and signalling
pathways (for review see Renard et al. 2007; Fig. 3).
Until now, following a similar approach to primate ESC
isolation, most attempts to culture bESC have been
inspired by the original culture methods for mESC of
Evans and Kaufman (1981).
Bovine ESCs are usually cultured on mouse embry-
onic fibroblasts (primary MEF or transformed STO
cells). Culture media consists of Dulbecco’s Modified
Eagle’s Medium supplemented with foetal bovine serum,
L-glutamine, 2-bmercaptoethanol and different growth
factors, mostly leukaemia inhibitory factor (LIF) and
epidermal growth factor (EGF) (for comparison of some
bESC culture conditions see Table 1).
Yet it is likely that culture conditions suitable to
maintain mESC could be inadequate to maintain
undifferentiated bESC. Preliminary studies by Keefer
(2007) showed that the bovine ICM and its primary
outgrowths express the LIF receptor and gp130 trans-
ducer. Yet, LIF did not improve the establishment and
maintenance of ESCs from other ungulates (see Vack-
ova et al. 2007 for review) although its presence in pig
ESC culture medium prevented EB formation (Brevini
et al. 2007). It can be speculated that, such as in hESC,
stimulation of the STAT3 pathway by LIF might not
induce proliferation of ungulate ESCs. Similarly, some
growth factors found to suppress differentiation of
mESCs [such as TGB-b, EGF or insulin-like growth
factors (IGFs)] did not inhibit differentiation of porcine
Bovine blastocyst
Oct-4/OCT-4
Murine blastocyst
Human blastocyst
NANOG/NANOG
OCT-4/OCT-4
NANOG/NANOG
Nanog/NANOG
OCT-4/OCT-4
NANOG/NANOG
Murine ESCHuman ESC
N
Bovine ESC
NANOG
OCT-4
Nanog
Oct-4
NANOG
OCT-4
OCT-4/OCT-4 OCT-4/OCT-4
Fig. 2. Expression of oct-4 and
nanog mRNA and proteins in
bovine, human and murine
blastocyst and embryonic stem
cells. Data from bovine are from
Kirchhof et al. (2000), Degrelle
et al. (2005), Gjørret and Maddox-
Hyttel (2005), Wang et al. (2005)
and Mun
˜oz et al. (2008). Data
from human are from Adjaye et al.
(2005), Cauffman et al. (2005),
Chambers et al. (2003) and Kimber
et al. (2008). Data from mouse are
from Palmieri et al. (1994), Kirch-
hof et al. (2000), Chambers et al.
(2003) and Hatano et al. (2005)
34 M Mun
˜oz, C Dı
´ez, JN Caaman
˜o, A Jouneau, I Hue and E Go
´mez
2008 The Authors. Journal compilation 2008 Blackwell Verlag
stem cells (Hochereau-de Reviers and Perreau 1993;
Prelle et al. 1994).
The inability of currently used culture conditions to
meet the nutrient or growth factor requirements of bESC
is one of the possible explanations for the failure to
isolate these cells. Identification of specific pluripotency
signalling pathways will help to determine which growth
factors are beneficial or which ones are inappropriate for
establishing a successful bESC culture.
In Vitro and In Vivo Differentiation of bESC
The differentiation ability of bESC in vitro is evaluated
by EB formation. EBs are aggregates of stem cells whose
development is reminiscent of early embryogenesis.
Maintenance of bESC in a suspension culture (Strel-
chenko 1996) or in the absence of a feeder layer (Saito
et al. 2003; Wang et al. 2005) initiates the formation of
EBs. The EBs are composed of two layers of cells,
ectoderm-like cells covered by a thin layer of endoderm
like cells, with heterogeneous cellular particles within the
cavity. The cells of bovine EBs give rise to a wide variety
of differentiated cell types, including derivatives of the
three germ layers (Saito et al. 2003; Wang et al. 2005).
This ability is a proof of their pluripotent-differentiation
character in vitro.
The ability of bESC to participate in the embryogen-
esis has been proven only after a short propagation
period in vitro. Chimeric transgenic calves have been
born after injection of bESC (passage 3) into cleavage
stage embryos (Cibelli et al. 1998), embryo aggregation
with bESC (passage 9–13) (Iwasaki et al. 2000). Yet
integration of ESCs into the germ line, one of the
properties used to define ESCs, have not been achieved
in any of these experiments.
Calves were also successfully cloned using ES-like
cells as donor nuclei (short cultured ICMs or passage
14–18) (Sims and First 1994; Saito et al. 2003), but the
use of ESC in nuclear transfer can not be taken as a
proof of pluripotency as cloned animals have been
produced from fully differentiated somatic cell nuclei
(Kato et al. 2000; Wakayama and Yanagimachi 2001).
Conclusions
The above summarized data indicate that although it
is possible to isolate bESC, suitable conditions for
BMP4 LIF
Mouse/Human
Activin/Nodal TGFβFGF
2
BMP4 LIF
Smad 1/5/8 STAT3/5 Smad 2/3 MAPK/ERK STAT3
Pluripotency Pluripotency Pluripotency Differentation
Ungulates
FGF
2
LIF
No effects on ESCPositive effects on No
establishment/maintenance ESC proliferation
Human
Mouse
Fig. 3. Pluripotency and differen-
tiation mediating pathways in
murine, human and embryonic
stem cells. Data from murine and
human are from Renard et al.
(2007). Data from ungulates were
reviewed by Vackova et al. (2007)
Table 1. A selection of reports on the culture media employed to
derive bovine embryonic stem cells
Reference Feeder layer Cell culture media
Talbot et al. (1995) Murine STO cells DMEM-M 199
FBS
b-ME
L-Glutamine
Non-essential
amino acids
Nucleosides
Mitalipova et al. (2001) Murine embryonic
fibroblasts
Alpha-MEM
FBS
b-ME
L-Glutamine
Saito et al. (2003) Murine STO cells MEM
FBS
b-ME
hLIF
hEGF
Wang et al. (2005) Murine embryonic
fibroblasts
Knock-out DMEM
FBS
b-ME
L-Glutamine
Non-essential
amino acids
hLIF
bFGF
Gjørret and Maddox-Hyttel
(2005)
Murine STO cells Knock-out DMEM
FCS
b-ME
L-Glutamine
MEM amino acids
hLIF
bFGF
Mun
˜oz et al. (2008) Bovine embryonic
fibroblasts
DMEM
FCS
MEM non-essential
amino acids
bFGF
bFGF, basic fibroblast growth factor; b-ME, b-mercaptoethanol; DMEM,
Dulbecco’s Modified Eagle’s Medium; FBS, foetal bovine serum; FCS, foetal
calf serum; hEGF, human epidermal growth factor; hLIF, human leukaemia
inhibitory factor; MEM, Minimum Essential Medium.
ESCs in Cattle 35
2008 The Authors. Journal compilation 2008 Blackwell Verlag
preventing spontaneous differentiation and senescence of
these cells are yet to be established. Similarly, validated
ESC lines have not been produced yet in other domestic
species (for review see Keefer et al. 2007).
Possible explanations for this failure are many,
including inadequate starting material or misleading
characterization of cell lines because of the inadequate
use of heterospecific markers. It is likely that the
inability to optimize culture conditions might have been
the biggest obstacle precluding success.
Until now empirical approaches, based almost exclu-
sively on protocols developed for derivation of mouse
stem cells, have been used to design cell culture environ-
ments for ES derivation in other species. Nevertheless,
taking in account the increasing differences reported in
the mechanisms and factors that regulate pluripotency
and self-renewal among mammals (for review see Renard
et al. 2007), it has become clear that alternative and ⁄or
complementary strategies will have to be applied.
Several techniques are currently used to identify the
gene expression profile of ESCs. These techniques include
serial analysis of gene expression, subtractive hybridiza-
tion, representational difference analysis, cDNA micro-
arrays and oligonucleotide microarray technologies. The
use of the above tools have allowed to identify unique
expression patterns of transcripts found in mouse and
human stem cells in comparison with differentiated
counterparts. The genes and pathways that appear to be
related to stemness in mouse and human ESCs are good
candidates to analyse basic mechanism governing pluri-
potency in mammalian ESCs. Furthermore, future tran-
scriptomic analysis among ESCs from different species
will help to identify species-specific pluripotency related
genes and pathways which could be used to design
tailored cell culture environments that enable ESC
derivation from other species.
Nuclear transfer could also provide an alternative
approach to improve ESC derivation (Renard et al.
2007). Studies with different strains of mice have shown
that the isolation of ESC can be hampered by the use of
different genotypes (Kawase et al. 1994). Thus in species
where the genetic background variability is high, the use
of genetically identical ICMs isolated from blastocysts
obtained by nuclear transfer could simplify the screening
for cell culture environments that would yield stable
ESC lines.
All the data gathered actually so far and that may be
collected in the near future about ESCs will help unravel
the mystery of the pluripotent state enabling the
establishment of ESCs from domestic animals.
Acknowledgements
Spanish Ministry of Science and Education (Project AGL2005–04479).
Dr M. Mun
˜oz is supported by a grant from FICYT.
Conflicts of interest
Authors of this paper have no conflicts of interest.
Author Contributions
All authors have contributed equally to write this paper.
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Submitted: 18 May 2008
Author’s address (for correspondence): Marta Mun
˜oz, Area de
Gene
´tica y Reproduccio
´n, SERIDA, Camino de los Claveles 604,
33203 Gijo
´n, Asturias, Spain. E-mail: mmunoz@serida.org
ESCs in Cattle 37
2008 The Authors. Journal compilation 2008 Blackwell Verlag
