Derivation and characterization of pluripotent embryonic germ cells in chicken

www.theriojournal.com

Theriogenology 67 (2007) 54–63

Derivation and characterization of pluripotent cell lines

from pig embryos of different origins

Tiziana A.L. Brevini, Valentina Tosetti, Mattia Crestan,Stefania Antonini, Fulvio Gandolfi *

Department of Anatomy of Domestic Animals, Centre for Stem Cell Research,

University of Milan, Via Celoria, 10, 20133 Milano, Italy

Abstract

Embryonic stem cells (ESCs) hold great promise for therapeutic use and represent a unique tool for investigating the process of

self-renewal and differentiation. The properties that make ESCs unique are their capacity of unlimited self-renewal coupled with the

property of re-entering the developmental process if returned inside a blastocyst. Such plasticity enable ESCs to form all embryonic

tissues including germ cells. However, these remarkable properties, at present, have been demonstrated only for mouse ESCs even if

cells with somehow more limited capacities have been derived in many different species including humans. The isolation of

pluripotent embryonic cells lines from human embryos marked a crucial change of perspective in evaluating the properties defining

an embryonic stem cell lines moving the focus from the generation of a germ-line chimera, obviously not feasible nor desirable in

human, to the capacity of these cells to differentiate both in vivo and in vitro in fully mature and functional cell types of all kinds.

Therefore, ESCs properties in species different from the mouse are being reassessed and re-evaluated, in view of their potential use

as experimental models for the development of clinical applications. Among the species that may play a useful role in this field,

the pig has a long-standing history as a prime animal model for pre-clinical biomedical applications and therefore, pig ESCs are

attracting renewed interest. In this review, we will summarize the current knowledge on this topic and will contrast the relatively

limited data available in this species with the much larger wealth of information available for mouse and human ESCs, in an attempt

to assess whether or not pig ESCs can actually become a useful tool in the fast growing field of cell therapy.

# 2006 Elsevier Inc. All rights reserved.

Keywords: Embryonic stem cells; Pluripotency; Cell plasticity; Nanog; Parthenogenesis

1. Why derive ESCs in farm animals and pig in

particular?

In 1981, it was demonstrated for the first time that the

isolation and culture invitro of the mouse blastocyst inner

cell mass originates stable cell lines, embryonic stem

cells (ESCs), that self renew indefinitely in vitro but, at

* Corresponding author. Tel.: +39 02 5031 7990;

fax: +39 02 5031 7980.

E-mail address: [email protected] (F. Gandolfi).

0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2006.09.019

the same time, retain their ability to differentiate in most

of the cell types that make up the adult individual [1,2].

Initially, these remarkable cells were mainly used as very

valuable models for studying the differentiation process.

Soon after, their capacity to integrate in a living embryo

forming germ-line chimeras combined with the proce-

dure of homologous recombination was used as a

powerful tool of genetic manipulation. The full realiza-

tion of their therapeutic potential only arrived when ESCs

were derived also from human embryos [3] making it

clear that these cells are perfect candidates for

regenerative medicine, tissue repair and gene therapy.

mailto:[email protected]

http://dx.doi.org/10.1016/j.theriogenology.2006.09.019

T.A.L. Brevini et al. / Theriogenology 67 (2007) 54–63 55

Nomenclature

CS calf serum

EB embryoid bodies

FB fibroblasts

FCS foetal calf serum

hLIF human recombinant leukocyte inhibitory

substance

hSC human recombinant steel factor

2ME b-mercaptoethanol

mLIF mouse recombinant leukemia inhibitory

substance

MEF mouse embryonic fibroblasts

NEAA non-essential amino acids

PEF pig embryonic fibroblasts

STO SIM mouse thioguanne and oubain resis-

tant fibroblasts

In recent years, reports have been appearing almost

daily in the scientific literature demonstrating that stem

cells can recapitulate embryonic and adult tissue

development, and can therefore repair injured or

congenitally defective tissues. However, there are

numerous hurdles that must be overcome on the way

to the routine application of ESCs as a therapeutic tool.

These include, but are not limited to, the need for

reliable ESCs differentiation protocols for different

lineages, purification techniques for the differentiated

progeny, as well as ways to circumvent the immuno-

logical rejection of transplanted cells. All this leads, in

the end, to a reliable assessment of how safe is to use

these cells in human patients.

Proof of principle experiments are now performed in

vitro both on mouse and human cell lines whereas in

vivo experiments are, for obvious reasons, limited to

mouse or other laboratory species. However, marked

morphological and physiological differences, a

reduced genetic variability and a short life span make

the mouse model rather unsatisfactory when experi-

mental outcomes need to be extrapolated to human

clinical applications. For this purpose, outbred large

animal models will be increasingly instrumental in the

safety and efficiency assessment of ESCs based

therapeutic methodologies. Among the possible spe-

cies, the pig has a long-standing tradition as a useful

and meaningful model in many branches of medicine.

Therefore, the derivation and characterization of

porcine ESCs has a huge potential as a privileged

experimental tool for the development of therapeutic

applications.

2. The role of species and pre-implantation

embryonic development in the process of ESCs

derivation

ESCs derive from a specific cell subpopulation of the

blastocyst named inner cell mass (ICM). It is also well

known that, at least in the mouse, ESCs can be

reintroduced into the ICM re-entering the process of

embryonic development [4]. However, the two cell

types, ESCs and ICM, are not equivalent since the ICM

exists only transiently in the embryo and does not act as

a stem cell compartment in vivo while ESCs form a

stable cell line in vitro. Therefore, ESCs are the result of

a selection and adaptation process to the culture

environment and, as such, could be considered more

an artifact than a physiological cell type [5]. If this is the

case, it has been suggested that the significant

differences observed between species in their capacity

to originate pluripotent cell lines from early embryos

may depend mainly on their ICM ability to adapt to an

arbitrary set of artificial conditions [6]. Indeed the

standard protocols for deriving mouse ESCs (mESCs)

work efficiently only with the 129 strain and, to a lower

extent, C57BL/6 strain [7]. The same procedures are not

equally successful with blastocyst of different genetic

background [8], confirming that there is a strong genetic

component to ESCs derivation [6].

The comparison between the trascriptomes of mouse

and human ESCs (hESCs) leads to similar conclusions.

In fact, combining together the results of different

studies it appears that only 13–55% of transcripts

enriched in mouse ESCs are also enriched in human

lines. When comparison is performed among different

human ESCs lines, the overlap rises to 85–99% (see [9]

for detailed references). The substantial differences

determined between human and mouse ESCs expres-

sion profiles are shared with some important functional

differences between the cell lines of the two species. For

instance, while ESCs of both typically grow well in

presence of mouse embryonic fibroblasts, hESCs do not

require the presence of leukemia inhibitory factor (LIF)

to activate JAK-STAT3 transcription factors in order to

maintain their pluripotency in culture. ESCs of both

species can be maintained in culture without a feeder

layer but in these conditions, hESCs require the

presence of fibroblast growth factor-2, whereas mESCs

need LIF and bone morphogenetic protein-4. By

contrast, BMP4 induces trophoblast differentiation in

hESCs.

Since genetic background has such a strong influence

on the initial frequency of establishing ESCs and on

their subsequent stability in culture, differences

T.A.L. Brevini et al. / Theriogenology 67 (2007) 54–6356

between species may be caused by the impact of genetic

heterogeneity typical of human and large animal species

that is completely absent in mouse ESCs.

Finally early mouse differentiation processes includ-

ing gastrulation, primary tissue specification and

organogenesis take place over approximately 4 days

and are well described. The corresponding processes in

human and other species span along 2–5 weeks after

fertilization, are inaccessible and very little is known

concerning their biology, which is likely to make more

difficult the production of differentiated cell types in

vitro.

At present, no data are available on comparisons

between human or mouse ESCs and cell lines of other

species. This would be very important in order to

determine whether or not alternative experimental

model resemble human lines more closely than murine

ESCs.

3. Main characteristics of pig pre-implantation

and differences with mouse and human

Detailed studies in mouse embryos determined that

the first differentiation process occurs at the late

morula stage when the outer cells adopt an epithelial

structure. This event is followed by the first appearance

of the blastocoel, which marks the divergence of the

first two lineages: trophectoderm and inner cell mass.

Upon blastocyst expansion differentiation continues

with the ICM forming two further cell lineages: the

epiblast and the primitive endoderm or hypoblast.

Between days 3.5 and 4.5 the epiblast will give rise to

the embryo itself and the hypoblast will evolve in the

extraembryonic endoderm and later will contribute to

the yolk sac. In other species these events take place

over a longer period of time. Although the three early

embryonic lineages are present in all eutherian

mammals the time between their formation and

fertilization is substantially shorter in mouse and

human than in domestic ungulates. Detailed studies in

mice have established that ESCs are derivatives of the

epiblast [10]. In humans, blastocyst formation of the

three early lineages takes approximately 6 days [11]

whereas in pig embryos epiblast formation begins at

hatching and is complete towards day 12 [12]. In

practical terms this translates in no defined epiblast

being present in pig blastocysts before hatching which

in vivo occurs on late day 6 or 7 of development [13].

At this stage the ICM is present, defined as the cells

comprised between the Rauber’s layer, separating it

from the uterine lumen, and the visceral endoderm.

Which is to say that, in order to obtain in vivo produced

pig blastocysts equivalent to their murine counterparts,

the uterus must be flushed on day 7 or 8 when embryos

are completely hatched and expanded [12]. Since

implantation in pig occurs around day 17, blastocysts

at later stages can easily be collected and eventually

used for ESCs derivation. However it must be noted

that, in pigs, a large variation in size is observed in

embryos collected at the same time and embryos of the

same size show substantial differences in the devel-

opment of the embryo proper. This means that when

post-hatching embryos are used for ESCs derivation,

the simple indication of the day of collection may

comprise a wide range of development. Upon hatching,

pig blastocysts retain a round shape and during days 8

and 9 the hypoblast is formed from the ICM and

gradually proliferates as a confluent cell layer

surrounding the blastocoel cavity. At the same time

and through day 10 the polar trophectoderm that covers

the epiblast, referred to as Rauber’s layer, begins to

degenerate until is completely lost leaving the epiblast

directly exposed to the uterine lumen [14]. The area

where the epiblast has surfaced is spherical, has a

whitish color under the stereomicroscope and is

referred to as the embryonic disc whose internal

surface is covered by cuboidal hypoblast cells.

Between days 11 and 13 development continues with

the embryonic disk and the whole conceptus begins to

assume an ovoid shape. At this stage the first signs of

polarity become visible under the stereomicroscope, in

the form of a crescent-shaped thickening within the

posterior third. This thickening originates within a

couple of days the rimitive streak which accompanies

the appearance of defined mesoderm and endoderm

layers. This differs from the mouse where mesoderm

and endoderm differentiation follows rather than

precedes the primitive streak formation [15]. Around

days 13 and 14, while the primitive streak is still

clearly visible, the major part of the epiblast trans-

forms into neural ectoderm and forms the neural plate.

This corresponds to a gradual down regulation of

typical pluripotency maker Oct4 which is substituted

by b-tubulin III expression, a marker of neural

differentiation. Therefore, it can be assumed that

embryos at this stage are no longer suitable for ESCs

derivation.

In summary, the extended pre-implantation period

together with the formation of an embryonic disk makes

pig embryo epiblast available for a much longer

time than in rodents and primates. Therefore, the

ICM available in the pre-hatching pig blastocyst

may not be exactly equivalent to the mouse epiblast

usually recovered for ESCs derivation. Differences in


pre-attachment development may be another factor

added to genetic variation explaining specie-specific

differences in ESCs derivation efficiency and pluripo-

tency.

4. The history of pig ESCs

The amount of data on pig ESCs is minimal when

compared to its mouse and human counterparts and is

schematically summarized in Tables 1 and 2. None of

the cell lines described in the literature satisfy all the

criteria required for a formally correct definition of

embryonic stem cells based on mouse ESCs standards

(like generation of germ-line chimeras, formation of

teratomas, stable karyotype, etc.) they should be defined

as stem cell-like or other dubitative descriptions.

However, since is the case for every other species

including human, and even if we are aware of such

limitations, for simplicity and clarity we prefer to use

the term embryonic stem cell.

The earliest attempts to derive pig ESCs were

published in 1990 [16–20]. Embryos were produced in

vivo by flushing the uterus between days 7 and 10,

therefore blastocyst were already hatched. Plating the

whole embryo was the most common approach [17,18]

but ICM isolation by immunosurgery was also performed

[20]. Based on the protocols used for the derivation of

mouse ESCs lines, DMEM was initially the basic culture

medium and this has remained substantially unchanged

up to now. Bovine serum of both fetal and adult origin has

always supplemented the medium together with b-

mercaptoethanol. Cell lines were propagated on STO

cells, the commonly used murine embryonic fibroblast

cell line used for mouse ESCs. Initially, insulin was the

only defined growth factor supplement investigated as

possible factor stimulating ESCs self renewal, whereas

the cytokine leukemia inhibitory factor was added later

[21] but with no evident effect. Morphology was the main

defining criteria for these ESCs. Most colonies were

described as ‘‘ES-like’’ when cells ‘‘were small and

rounded and had a large nucleus with one or two

prominent nucleoli’’ [20]. Beside this type of cell lines

other where derived with an ‘‘epithelial-like’’ appearance

described as cell with ‘‘flattened cuboidal shape and,

when grown to confluence, tended to form structures

reminiscent of epithelial sheets’’ [20]. These morpholo-

gical criteria were contradictorily linked to other

parameters of self-renewal and pluripotency. Epithe-

lial-like cell lines, in fact, were able to survive for a

number of passages higher than for the ES-like line.

Moreover, epithelial-like cells were able to form

embryoid bodies if cultured in suspension as opposed

to the ES-like line that did not show signs of

differentiation even after 30 days of the same culture

conditions [19,20]. Somewhat surprising was the

relationship between the two cell line morphologies,

with ES-like cells emerging from epithelial like colonies

even after four passages [19]. In other attempts, ES-like

cells gave rise to different morphologies including

endoderm-like and neural rosette [21], confirming how

difficult it is to relate cell line morphology with their

replication and differentiation potential. For this reason,

it is important to determine cellular and molecular

marker for defining the state of a cell line and to relate it to

the marker expressed by the epiblast in vivo. A first

important step towards this end are the results of Talbot

et al. who were the first to demonstrate alkaline

phosphatase as a reliable marker for undifferentiated

ESCs in pig and sheep [22]. This was followed by the

demonstration of a very limited stage-specific embryonic

antigen-1 (SSEA-1) expression [23] and by the lack of

expression of laminin, or of intermediate filaments like

vimentin and cytokeratins 8/18 which are absent in the

ICM and epiblast but detectable once differentiation

occurs [19,23,24].

Given the few molecular markers available for

characterizing the undifferentiated nature of the cell

lines, much effort was devoted to obtaining chimeras by

injecting putative ESCs into early embryos. This proved

to be a difficult achievement. Initially, it was possible to

obtain the birth of four chimeric piglets only when

freshly isolated ICM cells were injected into blas-

tocysts, with germ line chimerism demonstrated for the

first time in pig [25]. However, even a short period in

culture was able to prevent any cell integration. Around

the same time, pig chimeras were obtained by another

group injecting cells derived from a stable ESCs line

[26]. Unfortunately, no germ line transmission was

obtained in this set of experiments. Further attempts to

successfully produce chimeras from ESCs are rare, with

most studies unsuccessful and only a single chimera has

been described [27] with no germ line transmission. The

birth of chimeric piglet was also obtained injecting stem

cells derived from primordial germ cells but no germ

line transmission was observed in these cases [28–31].

This leaves mouse and chicken [32] as the only species

where germ line transmission has been described

following ESCs injection, although the latter were

passaged for a maximum of only three times.

As opposed to mouse ESCs, the most common way

to test pluripotency of human ESCs is injecting

undifferentiated cells into immuno-deprived mice and

to demonstrate the formation of teratomas, benign

tumors formed by several differentiated tissues. This

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Table 1

Summary of the main results describing ESCs derivation from pig embryos

Embryo origin d.p.i. ICM isolation method Feeder later Culture medium Maximum

passage

number

Method

undiffern ation

evaluatio

Method of evaluation and type

of differentiation

Reference

Ex vivo 7 or 9 Mechanical STO DMEM, 5% FCS, 10% CS, 0.1 mM 2ME >1 year >50

passage

Morphol y,

vimentin

negative

Morphology, muscle, FB, nerve,

endoderm

[17]

Ex vivo 10–11 Not specified Pig uterine cells

and STO

DMEM high glucose 10% FCS, 10% CS,

0.1 mM 2ME, NEAA, nucleosides,

0.2 mg/ml insulin

5 Morphol y Not reported [18]

Ex vivo 7–8 Whole embryo or

Immuno-surgery

STO or PEF DMEM, 10% FCS, 10% CS, 0.1 mM 2ME, LIF >10 Morphol y EB, citokeratin 18, vimentin,

no tumors, no chimera

[19,20]

Ex vivo 7–8 Immuno-surgery +

mechanical

dissection

STO DMEM or DMEM:199 15% FCS, 5% CS,

10–20 ng/ml hLIF

>80 passages

2 male lines

Morphol y Morphology, neuron, muscle,

epithelium, adipocytes, melanocytes,

glandular epithelim

[21]

Ex vivo 5–6 or 10–11 Whole embryo or

mechanical

dissection

PEF DMEM 10%FCS, 0.1 mM 2ME, 1000U/ml hLIF,

10 mg/ml hSCF, 10 ng/ml EGF, 4–25 ng/ml PDGF,

0,3 ng/ml TGF-b

11 passages Morphol y EB, Teratomas, [33]

Ex vivo 6–10 Immuno-surgery STO DMEM, 5% FCS, 10% CS, 0.1mM 2ME 10 passages Morphol y Morphology: epithelium, cardiac

muscle, FB, no chimera formation

[25]

Ex vivo 5.5–7.5 Whole embryo STO followed by

BRL condi-tioned

medium

DMEM high glucose, 0.1 mM 2ME, NEAA,

nucleosides, 20% FCS

44 passages Morphol y Morphology: FB adi-pocytes,

epithelium, neurons, muscles

cells,. EB, chimera

[26,53]

Ex vivo 7 or 11 Immuno-surgery

for day 7,

mechanical

for day 11

PEF DMEM 10%FCS 0.1 mM 2ME,

1000 U/ml hLIF

2 passages in

few days

SSEA-1

(in less n 5%

of the ce s, only

at attach ent)

Laminin, citokeratins 8/18 [23]

Ex vivo 7, unhatched

to very large

hat-ched >2 mm

Immuno-surgery Gelatine DMEM high glucose, 15%FCS

0.1 mM 2ME, or DMEM:owglucose:

HAMF10, 5% FCS 0.1mM 2ME,

NEAA, +/� LIF

4 days no

passages

AP Morphology, cytokeratin 8.13 [24]

Ex vivo 6–8 Whole embryo or

Immuno-surgery

STO ESC culture medium >35 passages 3

female lines 1

male line

AP Morphology showing polarized

epithelium EB, chimera

[27]

In Vitro 7–8 hatched Whole embryo STO for isolation,

gelatine for

maintenance

BRLconditioned DMEM 20%FCS,

2000 IU/ml LIF 10 ng/ml bFGF,

0.1 mM 2ME, NEAA

1 line 30

passages

Morphol y

epithelia ike

Used for nuclear transfer

with embryo development to

blastocyst stage

[42]

Ex vivo minipig 7–9 Whole embryo or

enzyme digestion

MEF DMEM 0.1mM 2ME, NEAA, 40 ng/ml hLIF,

20 ng/ml bFGF, nucleosides, 16% FCS

6 passages Morphol y, AP Morphology: neuron, smooth muscle,

epithelial EB

[36]

Ex vivo minipig 7–9 Enzyme digestion or

immuno-surgery

MEF, PEF, STO DMEM 0.1mM 2ME, NEAA, 40 ng/ml hLIF,


9 passages Morphol y, AP Morphology, neuron,

smooth muscle, epithelial

[38]

In vitro 4-c BL Whole embryo MFF DMEM 0.1mM 2ME, NEAA, 40 ng/ml hLIF,


4 passages Morphol y, AP EB Morphology, FB, neurons, EB [37]

In vitro

partheno-genesis

6 Immuno-surgery STO Low glucose DMEM:Ham F10 medium,

1000 IU/ml mLIF, 5% FCS, 10% KO

serum replacement

32 passages Oct4, N og,

SSEA-4 P,

TRA1-8

TRA 2-5

Morphology, vimentin,

cytocheratin, desmin, a-amilase,

BMP-4, neurofilament

[45],

unpublished

observations

Ex vivo minipigs 6 Immuno-surgery STO Low glucose DMEM:Ham F10 medium,

1000 IU/ml mLIF, 5% FCS, 10% KO

serum replacement

11 passages Oct4, N og,

SSEA-4 P,

TRA1-8

TRA 2-5

Morphology, vimentin,

cytocheratin, desmin,

a-amilase, BMP-4, neurofilament

[44]

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og

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og

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Table 2

Derivation efficiency of ESCs colonies

Embryo age (days) Colonies/embryo Efficiency

(%)

Reference

4-cell-MO in vitro 0/73 0 [37]

5–6 in vitro 2/12 16.5 [37]

6 in vitro 28/155 18 [44,45]

5–6 1/8 12.5 [33]

6–7 0/194 0 [25]

5.5–7.5 11/214 5 [53]

6–8 12/56 21 [27]

7–8 11/174 6 [19]

7–8 7/54 13 [21]

7–9 19/28 68 [36]

7–9 24/32 75 [38]

8–10 4/842 0.5 [25]

10–11 14/41 34 [18]

10.5–11 10/24 42 [33]

No obvious effect of embryo age is evident.

alternative has been little explored with porcine ESCs

and only in one case the formation of teratomas was

described [33]. In this case both days 5–6 and 10–11

blastocysts were used to derive ESCs but only cells

derived from the older embryos formed tumors when

transplanted in nude mice. Indeed other attempts with

ESCs lines derived from days 7–8 embryos were

performed but failed [19]. However, the relative

difficulty in obtaining teratomas from the earlier stages

of pig embryonic development was confirmed by

Anderson et al. [25], who described how prompt

teratoma formation was obtainable only by injection of

days 11 and 12 blastocysts under the kidney capsule,

whereas earlier stages rarely formed tumors. It is

interesting to observe that no cases of undifferentiated

teratocarcinomas were obtained, suggesting that

embryos of these stages are unsuitable for the derivation

of pluripotent embryonal carcinoma cell lines resem-

bling those obtained in mouse. Indeed, similar

observations were also reported for bovine embryos

[34], suggesting that ungulate embryos in general are

not amenable to this kind of manipulation in order to

obtain undifferentiated pluripotent cell lines.

The generation of embryoid bodies (EB) is an

alternative in vitro approach to test ESCs plasticity.

Embryoid bodies are aggregates of stem cells, the

development of which is reminiscent of early embry-

ogenesis and are generally considered as a positive

indication of stem cell plasticity [35]. Moreover, they

represent a common intermediate step for the derivation

of differentiated cell types [35]. As opposed to chimeras

and teratomas, embryoid body formation from pig ESCs

has been reported more frequently [19,20,26,36,37].

However, a clear correlation between cell line

morphology and EB formation was not always possible

[20], with ES-like cells unable to differentiate into

cystic EB, whereas epithelial-like lines were readily

able to form these structures upon suspension culture.

In addition, or as an alternative, to EB formation,

further in vitro characterization of cell plasticity has

been determined in many cases by using morphological

criteria, and several cell lines were described as capable

of differentiation into different cell types including

fibroblasts, neurons, adypocites, muscle cells, epithelial

cells [16,19,21,26,36–38]. However, three very detailed

studies convincingly demonstrate that it is possible to

derive liver bile ductules [39], neurons, astrocytes [40]

and macrophages [41] from pig ESCs. These results are

quite remarkable, as ES-derived macrophages show

specific morphology, at the microscopic ultrastructural

level, reactivity with cell-specific monoclonal anti-

bodies, phagocytic behavior and formation of phago-

somes as well as cytotoxic activity [41]. Differentiation

towards the neural lineages was spontaneous and was

demonstrated by strict morphological criteria, together

with the expression of specific neural cell markers such

as GFAP and vimentin. Biliary epithelium was induced

by culturing pig ESCs at pH 7.6–7.8 thus obtaining

secreting ductules responsive to a series of challenges in

vitro [39]. Taken together, these three reports demon-

strate that pig ESCs were able to differentiate in vitro

into cells representative of endodermal, neuroectoder-

mal and mesodermal origins and were therefore

pluripotent.

All the results described above were obtained with in

vivo produced embryos. Only recently has the deriva-

tion of ESCs line from in vitro generated pig blastocyst

been reported. In the first report, hatched blastocysts

cultured in vitro for 7–8 days were used [42] and one

cell line of epithelial-like morphology was obtained.

The line was stable for up to 30 passages and when

individual cells were used as the donor cell for nuclear

transfer experiments, embryos reached the blastocyst

stage [42]. More recently, the ability to form ESCs lines

of in vitro generated embryos ranging from 4-cell to the

non-hatched blastocyst stage was examined. The results

indicated that ESCs can be obtained only upon

blastocyst formation [37] and even this result requires

cautious interpretation, since the three lines described

were maintained for only four passages.

5. Some recent characterizations and innovativederivation

The results described above indicate that although it

is possible to derive ESCs lines from pig embryos, their


characterization has been based mainly on morpholo-

gical criteria and, up till now, have yet to take advantage

of the detailed molecular knowledge that has been

accumulating over the years in mouse and human ESCs.

It is now well established in mouse and human ESCs

that two genes are the main hallmarks of pluripotency:

Oct4 and Nanog. The relationship between the two is

unclear but some evidences suggest that an Oct4

binding site is present in the Nanog upstream sequence.

The pattern of Oct4 expression has been well

characterized in the pig embryo. The protein is present

in the oocyte and remains visible throughout the early

cleavages and blastocyst formation. This differs to

what is observed for mouse embryos, where Oct4

expression is down regulated in the trophectoderm, in

pig embryos a strong Oct4 signal is visible in all cell

types present in the blastocyst until hatching from the

zona pellucida takes place [43]. At this stage a

restriction of Oct4 expression is detected as in the

mouse and the transcription factor can be demonstrated

exclusively in the epiblast component of the embryonic

disc [12]. We have recently derived pluripotent pig

lines from both in vivo [44] and in vitro [45] embryos

and we followed Oct4 expression during the life of the

cell lines. In our experimental system we used zona

enclosed blastocyst and ICM was isolated by immu-

nosurgery before plating them on a feeder layer of

inactivated STO cells. Our results indicate that Oct4 is

expressed at the time of attachment and during the first

passages for a maximum of seven when its expression

becomes completely down regulated. However despite

the lack of Oct4 expression, cells could be cultured for

Fig. 1. Comparison of the sequence obtained porcine Nanog cDNA with the

base. Sequence of these species have been derived from EMBL and GenBan

Mus musculus, AY278951; Capra hircus, AY786447.

several months without any overt change in morphol-

ogy and without up regulation of specific differentia-

tion markers. This suggests that other factors may be

responsible for the maintenance of the self-renewal of

these cells in an undifferentiated status. For this reason

we investigated the expression of Nanog, which

together with Oct4, is known to characterize human

and mouse pluripotent cell lines [9] and to determine

the epiblast fate of undifferentiated mouse ICM cells

[46]. A partial sequence of the Nanog gene (EMBL

AJ877915) was determined from pig somatic tissues

and embryos showing high homology with both the

human (84%), mouse (97%) and goat (82%) homo-

logues (Fig. 1). The gene is expressed in some adult

somatic tissues (ovary, heart and muscle) but not in

others (spleen, lung and brain) [47], which is a more

restricted organ distribution difference compared to

the mouse ([48], Fig. 2). In contrast, the embryonic

expression pattern during early development to the

pre-hatching blastocyst stage follows the same pattern

in the two species, since in both cases Nanog

expression appears after genome activation ([48],

Fig. 2). Using this background information, we

analyzed Nanog expression in our pig cell lines and

determined that this gene is always expressed even in

the absence of Oct4 (Fig. 3). This indicates that pig

ESCs self-renewal can occur upon Nanog expression

alone without the strict requirement of the contem-

porary expression of Oct4. This is consistent with

recent reports indicating that Nanog over expression is

sufficient for maintaining mouse ESCs in the undiffer-

entiated state [49].

human, mouse and goat orthologs. Asterisks (*) indicate the conserve

k under the following accession numbers: Homo sapiens, AB093576;


Fig. 2. (A) Expression of Nanog in porcine oocytes and pre-implantation embryos. b-Actin was used as an internal control for RT-PCR Reaction. (B)

Expression of Nanog in porcine tissues. b-Actin was used as an internal control for RT-PCR reaction.

Fig. 3. Expression of Nanog in pig ESC colony.

Despite the unusual expression pattern of Oct4, Nanog

expression is well correlated with an undifferentiated

state. In fact, if cultured with the hanging drop method

our pig cells will form embryoid bodies which undergo a

rapid growth and cavitate. These structures support

cellular differentiation as indicated by the expression of

a range of genes markers for derivatives of the three

germ layers, while Nanog expression is down regulated.

Finally, we used the pig model to investigate the

possibility of deriving stem cells from parthenogenetic

embryos. The scope of such work was to develop

models of ESCs that do not require the sacrifice of

viable embryos, since this aspect raises serious ethical

concerns when applied to human embryos and the

generation of human cell lines. Parthenotes are the

product of oocyte activation, without the intervention of

a sperm cell. They follow a developmental process

almost identical to that of a normal embryo but they

cease to develop shortly after implantation due to lethal

epigenetic alterations. The possibility to obtain parthe-

nogenetic ESCs has already been demonstrated in

mouse [50] and Cynologous monkey [51] raising

considerable interest in view of its possible application

to the human species.

We obtained pig parthenogenetic blastocyst follow-

ing a protocol based on the sequential exposure of the

oocyte to the calcium ionophore, ionomycin, followed

by the protein synthesis inhibitor, 6-DMAP, in order to

prevent the extrusion of the second polar body. Diploid

embryos are usually obtained that form blastocyst with,

on average, 76 blastomers and a well-defined ICM [52].

As for the in vivo embryos, the zona pellucida was

removed enzymatically and the ICM was isolated

immunosurgically. The support of a STO feeder layer

was used to facilitate the attachment of the isolated ICM

which began to proliferate. At present two cell lines

have been maintained in culture with no sign of

differentiation for more than 2 years. Cells were able to

resume their growth in culture after two rounds of

freezing and thawing. Upon feeder layer removal the

cell began to spontaneously differentiate into neural and

other cell types [45].

6. Conclusions

Pluripotent cell lines can be isolated from pig

embryos obtained in vivo, in vitro and by parthenogen-

esis. The procedures follow closely those developed for


mouse and human cell lines and, recently, can take

advantage of the wealth of specialized reagents that

followed the isolation and extensive research on human

ESCs. Most cell lines were characterized mainly on

morphological criteria in the absence of specie specific

molecular probes, however more recently such tools

have been developed and can provide more detailed

insight on the properties of these cells. The work in

progress is expected to rapidly standardize and optimize

derivation and culture protocols for this species

enabling the development of interesting experimental

models that will prove to be valuable in the progress of

human cell therapy

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Derivation and characterization of pluripotent embryonic germ cells in chicken

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