CHARACTERIZATION OF PORCINE TROPHECTODERM DERIVED CELLS TO

ESTABLISH A MODEL FOR PLACENTAL DEVELOPMENT

by

KATHRYN STEWART HODGES

(Under the Direction of Steven L. Stice)

ABSTRACT

A porcine trophoblastic cell line could provide a powerful model for understanding

trophoblast cell biology as well as placental gene expression and proteomics in vitro. Bone

morphogenetic protein 4 (BMP4) has been shown to induce differentiation of human embryonic

stem cells into trophoblast lineages. In this experiment, elongated embryos were flushed from the

hysterectomized uteri of superovulated and bred prepuberal gilts 15 days post insemination. The

trophectoderm tissue was manually dissected into cell aggregates and plated on collagen Type

IV, Matrigel, human extracellular matrix (laminin, collagen type IV and heparan sulfate

proteoglycan derived from human placenta) or mouse embryonic fibroblast (MEF) feeder layers

in a DMEM based culture medium in the presence or absence of BMP4. Cells were passaged

and only cells growing on Matrigel or MEF feeder layers could be further cultured. These data

suggest that both the cell substrate and BMP4 affect initial trophoblast outgrowths.

INDEX WORDS: Porcine, Placenta, Trophoblast, Bone morphogenetic protein 4, Somatic

cell nuclear transfer

CHARACTERIZATION OF PORCINE TROPHECTODERM DERIVED CELLS TO

ESTABLISH A MODEL FOR PLACENTAL DEVELOPMENT

by

KATHRYN STEWART HODGES

B.S.A, University of Georgia, 2001

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment

of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2004

© 2004

Kathryn Stewart Hodges

All Rights Reserved

CHARACTERIZATION OF PORCINE TROPHECTODERM DERIVED CELLS TO

ESTABLISH A MODEL FOR PLACENTAL DEVELOPMENT

by

KATHRYN STEWART HODGES

Major Professor: Steven L. Stice

Committee: Clifton A. Baile Scott L. Pratt

Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2004

iv

DEDICATION

To my husband Will, who has made it all worthwhile.

v

ACKNOWLEDGEMENTS

The author would like to begin by thanking Dr. Steven L. Stice, chairman of the

supervisory committee and mentor. His guidance and direction throughout the graduate school

program and the writing of this thesis have been greatly appreciated. Also thanks go to the rest of

the advisory committee, Dr. Clifton A. Baile and Dr. Scott L. Pratt, for their willingness to serve

on the graduate committee, thoughtful input, and for reviewing this thesis.

Thanks are also due to many who assisted in both farm and lab tasks. They include fellow

graduate students Allison Adams and Nanci Williams, who spent hours upon hours between the

Swine Center, LARU and the lab helping collect data, Mike Daniel at the Swine Center, for

providing valuable assistance with the pigs, and Randy Gabriel and the crew of LARU, for

aiding with the care of the animals used in this experiment. Dr. Maya Mitalipova provided

excellent advice on topics about manual passage and stem cells. Thanks are also expressed to

Dr. Steve Nickerson, department chair, and Dr. Mark Froetschel, graduate coordinator, for the

opportunity to study in the Animal and Dairy Science Department.

Special thanks are due to fellow graduate students and co-workers Allison Adams, Nanci

Williams, Emily Duff, John Gibbons, Eric Sherrer, Deanne Tibbetts, Christine Henderson, Gail

Palmarini, Debbie Hoyer, Soojung Shin, Pablo Bosch, Alison Venable, John Calhoun, Raj Rao

and others for their help, but more importantly for their friendship over the past two and a half

years.

Most importantly, the author would like to thank her family. None of this work would be

possible with out the love and support of her husband throughout the many tasks and decisions of

vi

graduate school. In addition, thanks to her parents, Robert and Martha Carol Stewart. Her father

has served as a role model in life as well as in academia and both parents have been encouraging

and supportive throughout this work. Thanks also go to the author’s brother and partner in

crime, Lawton, and his wife, Beth. Last, but not least, the author would like to thank her sister,

Sally.

vii

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS.............................................................................................................v

LIST OF FIGURES ....................................................................................................................... ix

CHAPTER

1 Introduction....................................................................................................................1

2 Literature Review...........................................................................................................4

Chorioallantoic Placentation .....................................................................................4

Development of the Porcine Blastocyst ....................................................................5

Maternal Recognition of Pregnancy..........................................................................6

Placental Development..............................................................................................7

Trophoblast Cells in Culture .....................................................................................9

Interferon-γ ..............................................................................................................11

Bone Morphogenetic Protein 4................................................................................13

Summary .................................................................................................................16

3 Characterization of Porcine Trophectoderm Derived Cells in the Presence or hBMP4

in the Absence of a Feeder Layer............................................................................17

Introduction .............................................................................................................17

Materials and Methods ............................................................................................19

Results .....................................................................................................................25

Discussion ...............................................................................................................37

viii

Conclusions .............................................................................................................40

REFERENCES........................................................................................................44

ix

LIST OF FIGURES

Page

Figure 1: Cascade of BMP signaling .............................................................................................15

Figure 2: Feeder-free cell culture...................................................................................................30

Figure 3: Feeder-free culture versus culture on a feeder layer ......................................................31

Figure 4: Phase microscopic images of TSC1 after 7 days in culture. ..........................................32

Figure 5: Phase microscopic images of TSC5 after 7 days in culture. ..........................................33

Figure 6: Phase contrast microscopic images of TSC5 after the first passage. .............................34

Figure 7: Interferon-γ gene expression, TSC5...............................................................................35

Figure 8: α-fetoprotein expression, TSC5......................................................................................36

Figure 9: Phase contrast microscopic image of Oil Red O staining in trophoblastic cells ............37

1

CHAPTER 1

INTRODUCTION

The 1997 announcement of Dolly, the first clone of an adult animal, introduced somatic

cell nuclear transfer (SCNT) as a promising candidate for applications in animal agriculture and

human medicine. For agriculture it marked the opportunity for the clonal expansion of

genetically superior food producing animals. Somatic cell nuclear transfer, in conjunction with

genetic modification, provides a means for studying a variety of developmental processes and

genetic disorders. Farm animals can become bioreactors producing limitless organs for

xenotransplantation or pharmaceuticals. The restoration of endangered or even extinct species is

yet another possibility. However, until the full potential of SCNT can be realized, researchers

must overcome several obstacles.

Although several diverse species have been cloned since Dolly, (Kato, Tani et al. 1998;

Wakayama, Perry et al. 1998; Baguisi, Behboodi et al. 1999; Polejaeva, Chen et al. 2000;

Chesne, Adenot et al. 2002; Shin, Kraemer et al. 2002; Galli, Lagutina et al. 2003) there are

major inefficiencies that limit the application of nuclear transfer technology. Dolly was the only

live offspring produced from 227 attempts resulting in a 0.4% efficiency rate (Wilmut, Schnieke

et al. 1997). Consistent among all published research is that only a small portion of embryos

reconstructed using adult cells developed to become live offspring with cloning efficiencies

ranging from 0 – 7.2% (Wilmut and Peterson 2002). This inefficiency makes the commercial

application of nuclear transfer difficult and expensive.

Numerous physical abnormalities have been associated with cloned progeny. Large

offspring syndrome is a common outcome of embryonic and somatic cell cloning in sheep and

cattle (Willadsen, Janzen et al. 1991; Wilson, Williams et al. 1995; Cibelli, Stice et al. 1998;

2

Wells, Misica et al. 1999) and has been postulated to be caused by the inadequate

reprogramming of the somatic cell nucleus by the oocyte (Bourc'his, Le Bourhis et al. 2001;

Dean, Santos et al. 2001; Kang, Koo et al. 2001; Ohgane, Wakayama et al. 2001; Archer, Dindot

et al. 2003). Genomic imprinting abnormalities in gene expression may induce increased fetal

growth in utero (Trounson 2001). Gene targeting studies in sheep have resulted in a high

occurrence of kidney defects and above normal liver and brain pathology (McCreath, Howcroft

et al. 2000). Cardiac and circulatory abnormalities, in addition to severe cases of hepatic

lipidosis, have also been reported in cloned sheep and cows (Hill, Roussel et al. 1999; McCreath,

Howcroft et al. 2000).

Placental abnormalities are a reoccurring theme in cloned offspring. Malformed

placentas are commonly reported in bovine pregnancies from cloned embryos, suggesting

problems in the extraembryonic lineages that may relate to genomic imprinting or other

epigenetic interference to gene expression patterns (Cibelli, Stice et al. 1998; Hill, Roussel et al.

1999; Wells, Misica et al. 1999). Hydrallantois and hydramnios are also observed in cloned

bovine pregnancies (Willadsen, Janzen et al. 1991; Cibelli, Stice et al. 1998; Lewis, Peura et al.

1998; Hill, Roussel et al. 1999). Other placental deformities in ruminate species include fewer

placentones, a significant increase in placentone size and enlarged umbilical structures (Stice,

Strelchenko et al. 1996; Hill, Roussel et al. 1999; Wells, Misica et al. 1999).

Ogura et al. (2001) reported that only 1.2-5.3% of cloned mouse embryos developed to

term, and upon examination of recipient uteri at days 9.5-11.5, 90% of the cloned embryos

transferred had died (Ogura, Inoue et al. 2002). Histological studies showed that 50% of cloned

embryos did not undergo chorionic plate formation, which implicates placental defects as a

major cause of death (Ogura, Inoue et al. 2002). Cloned mice that successfully developed to

3

term had placentas that were consistently two to three times larger than those of controls (Inoue,

Kohda et al. 2002; Ogura, Inoue et al. 2002). In addition, gene expression in those term

placentas exhibited a significant reduction in the expression of imprinted and nonimprinted genes

(Inoue, Kohda et al. 2002).

Further research is necessary to better understand the processes involved in nuclear

transfer and the reprogramming of development. Studies involving both in vitro and in vivo

development of the placenta are significant because abnormal placentation has been implicated

as a factor involved in nuclear transfer losses. To implement these studies, a feeder-free

trophoblast stem cell line should be developed to determine the molecular and cellular events in

normal and, in the case of cloned pregnancies, abnormal placental development.

The versatile range of placental structures across species, results in the hypothesis that

these anatomical and physiological differences may account for the differences in placental

abnormalities seen in offspring produced by nuclear transfer. Among the farm species, pigs have

the greatest potential in animal agriculture and human medical applications. Thus, the

establishment of a porcine trophoblast cell line would provide a powerful model for

understanding trophoblast cell biology as well as placental gene expression and proteomics in

vitro.

4

CHAPTER 2

LITERATURE REVIEW

Chorioallantoic Placentation

The mammalian placenta is often overlooked for the role it plays in development.

Bidirectional communication between the fetus and the mother is vital for a successful

pregnancy. It is feto-maternal contact by means of the placenta, a remarkably versatile

epithelium, that provides hormone production, specific nutrient absorption, selective transport,

active metabolism and the ability to resist maternal immunological attack. The integration of

these processes results in the production of live, healthy offspring (Wooding and Flint 1994).

Thus, proper formation of the placenta is crucial to successful reproduction.

The great diversity of placental structures within the eutherian mammals has led to many

attempts to devise a classification system that would categorize the placenta based on common

structural features. The first and simplest criterion is the shape of the term placenta (Wooding

and Flint 1994).

1. Diffuse: villi over entire surface, e.g. pig, horse.

2. Cotyledonary: chorionic villi grouped into a characteristic number of discrete

tufts which can vary from 5 (deer) to 150 (giraffe), e.g. ruminants.

3. Zonary: villi restricted to an equatorial band or patch, e.g. carnivores.

4. Discoid: villi restricted to a single or double disc, e.g. humans, rodents,

insectivores, anthropoids.

The second criterion is a structural classification based on the number of placental tissue

layers that separate the maternal and fetal blood circulation (Grosser 1927). Placentas are

5

grouped according to how many layers of maternal tissue were removed by the persistent chorion

during development. The result produces four placental categories (Wooding and Flint 1994).

1. Epitheliochorial: no layers removed; uterine epithelium in direct contact with the

chorion, e.g. pig, horse.

2. Syndesmochorial: uterine epithelium removed, maternal connective tissue in

contact with the chorion, e.g. ruminants.

3. Endotheliochorial: maternal uterine epithelium and connective tissue removed,

maternal endothelial basement membrane in contact with the chorion, e.g. carnivores.

4. Haemochorial: all maternal tissue layers removed, chorion bathed directly in

circulating maternal blood, e.g. rodents, insectivores, anthropoids.

The epitheliochorial placenta occurring in pigs consists of an apposition and attachment

of fetal membranes to the maternal endometrium after a long preimplantation period (Burghardt,

Bowen et al. 1997). Epitheliochorial placentation involves a vast increase in area with no loss of

layers between fetal and maternal bloodstreams. The exclusively cellular layers become

exceedingly attenuated, reducing the diffusion distance as pregnancy proceeds, but all persist to

parturition (Wooding and Flint 1994). Throughout gestation, the outermost fetal layer of the

placenta is invariably the trophoblast or a trophoblast derivative (Wooding and Flint 1994).

Development of the Porcine Blastocyst

The first cellular division of a fertilized porcine embryo occurs at 14-16 hours after

fertilization (Hunter 1974). The second cleavage begins 7 hours later (Hunter 1974). Further

cleavage divisions follow in rapid succession (Patten 1948). By Day 5, these cellular cleavages

produce a cluster of cells, the morula, still enclosed in the protective zona pellucida (Barends,

Stroband et al. 1989). At the 16-32 cell stage, compaction begins and the outermost cells of the

6

morula flatten against the zona pellucida (Marrable 1971). A cavity, the blastocoele, appears

within the core of the cluster forming an early blastocyst stage embryo. An internal mass of cells

is established at one pole. This inner cell mass (ICM) is destined to form the developing fetus.

The thin monolayer of cells that constitute the blastocyst wall are the trophoblast from which the

extraembryonic tissues will form. This distinction between the trophoblast and ICM represents

the first differentiation event in mammalian development (Patten 1948).

By Day 6 of development, the trophoblast and the ICM become readily distinguishable in

a spherical blastocyst (Albertini, 1987). Hatching occurs and the whole blastocyst now

undergoes a rapid expansion together with a radical change of shape (Barends, Stroband et al.

1989). The embryo changes in shape from a sphere 0.2 mm across at Day 6, through ovoid and

tubular intermediates to form a filamentous blastocyst, about 100 cm in length when unraveled

by Days 11-14 (Marrable 1971; Anderson 1978; Geisert, Brookbank et al. 1982). The growth of

the ICM is minor compared to that of the trophoblast. The trophoblast cells covering the ICM

(polar trophectoderm) disappear around Day 10 (Barends, Stroband et al. 1989). As a result, the

ICM forms part of the outer cell layer from this point forward (Barends, Stroband et al. 1989).

By Day 12 the embryonic mass appears as a round patch at or near the midpoint of the elongated

conceptus. The single layered trophoblast is now reinforced by endoderm cells emigrating from

the embryonic mass (Patten 1948; Marrable 1971). The cells that remain in the ICM are

rearranged in an orderly array of columnar cells now known as the embryonic disc (Marrable

1971).

Maternal recognition of pregnancy

In the bicornate uterus of the pig, blastocysts normally implant at evenly spaced sites

along both uterine horns (Anderson 1978; Wooding and Flint 1994). How this spacing is

7

achieved is unclear, but experimental observations indicate considerable capacity for blastocyst

migration along the uterine lumen (Dziuk 1968). The most likely explanation is a passive

movement propelled by vigorous contractions of the uterine musculature (Wooding and Flint

1994).

At Days 8-11, the pig conceptus is ovoid or tubular in shape and evenly spaced

throughout the uterus (Anderson 1978; Wooding and Flint 1994). Between Days 10 and 12, the

conceptuses begin to secrete estrogen (Bazer 1989) and an interferon like molecule (La

Bonnardiere 1993). A minimum of four conceptuses are necessary to stimulate the uterus to

modify its prostaglandin secretion (Wooding and Flint 1994). The uterine luteolysin in pigs is

prostaglandin, although the endocrine requirements for luteolysis in pigs has not been clearly

delineated (Bazer 1989). Estrogens produced by the conceptuses between Days 11 and 12 of

gestation provide the initial signal for the maternal recognition in swine (Geisert, Renegar et al.

1982). Prostaglandin-F2-alpha secretion transitions from an endocrine direction to an exocrine

direction between Days 10 and 12, which is temporally associated with estrogen secretion by the

elongating pig conceptus (Bazer 1989). Injection of exogenous estrogen on Days 11-15 of the

estrous cycle results in corpus luteum maintenance for a period slightly longer than gestation

(Conley 1989), confirming that estradiol indeed has a luteotropic effect in the pig.

Placental Development

The formation of the trophectoderm is the first clear sign of differentiation in early

embryonic development. The presence of intercellular junctional complexes in the outer cells of

the early compacting embryo is one of the most important morphological features of the

trophoblast (La Bonnardiere 1993). The permeability barrier established by these complexes

plays an essential role in the establishment of the microenvironment within the embryo, so that

8

the blastocoelic fluid can initially accumulate in the space between the cells and then induce

blastocyst expansion (Loke and Whyte 1983). Consequently, the trophoblast is a typical

polarized epithelial cell (La Bonnardiere 1993).

At implantation, in the pig, the conceptus is mostly a trilaminar blastocyst with the

endoderm cells and a briefly functional yolk sac lining the trophectoderm (Wooding and Flint

1994). Once elongated, the filamentous blastocyst interacts with the immediately underlying

uterine epithelium (Dantzer 1985). Using scanning and transmission electron microscopy,

Dantzer implicated several structures in the initial stages of placentation in the pig. At Day 13,

protruding epithelial proliferations on the uterine endometrium become visible. These

protrusions are more obvious on Day 14 and become enclosed by chorionic caps. This

interaction between the endometrial epithelium and the trophoblast immobilizes the blastocyst

and keeps maternal and fetal sides together so that cell-to-cell contact can develop. A thick

glycocalyx is evident on the maternal and a thin one on the fetal epithelium before close contact

is established. On Day 15, the fetal and maternal epithelia come into close apposition, involving

loss of microvilli and considerable glycocalyx erosion. As the fetal and maternal microvilli

reform, they now interdigitate increasing the feto-maternal exchange surface area by 10-12 times

over a flat apposition (Baur 1977). There is no indication for the formation of any junctions

between the fetal and maternal epithelia or for penetration of their tight junction apical seals by

cellular processes from either side (Spencer and Bazer 2004).

Unlike ruminant species, the porcine trophoblast, never erodes the uterine epithelium and

never gains direct access to the maternal bloodstreams (Geisert, Renegar et al. 1982). At 17

days, the proliferation of fetal trophectoderm over the gland mouths has started to form regular

areolae (Wooding and Flint 1994). Areolae are unique placental structures in ruminants and pigs

9

that develop over the mouth of each uterine gland as specialized areas for absorption and

transport of uterine histotroph (Spencer and Bazer 2004). About 7000 regular areolae per

porcine conceptus are present, fairly evenly spaced, from early gestation to parturition (Wooding

and Flint 1994). At 18-22 days post conception, the conceptus expands so that it touches the

epithelium around the entire circumference and establishes microvillar interdigitation over the

entire trophectoderm (Dantzer 1985).

The end result is maximized surface area available for exchange between the fetal and the

maternal capillaries (Wooding and Flint 1994). This is achieved in the pig by development of a

meshwork of capillaries just under the epithelium of each fetal and maternal villus (Dantzer

1985). As the conceptus grows, the feto-maternal junctional area and the transport capacity of

the placenta increase continuously until parturition (Wooding and Flint 1994).

Trophoblast cells in culture

The isolation of various bovine, ovine, porcine and caprine trophoblast cell lines with the

use of mouse embryonic fibroblast feeder layers have been described in several systems (Steven,

Mallon et al. 1980; Ramsoondar, Christopherson et al. 1993; Flechon, Laurie et al. 1995; Tanaka,

Kunath et al. 1998; Talbot, Caperna et al. 2000; Shimada, Nakano et al. 2001; Cencic and La

Bonnardiere 2002; Miyazaki, Imai et al. 2002). Investigators have shown that bovine trophoblast

cells (Shimada, Nakano et al. 2001), and more recently porcine trophoblast cells, can be

propagated without a feeder layer (Shimada, Nakano et al. 2001; Cencic and La Bonnardiere

2002).

Two approaches have been used to derive trophoblast cell lines. The first has been to use

the primary outgrowths from cultured blastocysts (Flechon, Laurie et al. 1995; Tanaka, Kunath et

al. 1998; Talbot, Caperna et al. 2000; Shimada, Nakano et al. 2001). The second method has

10

been to dissect the embryonic disk away from an elongated blastocyst and culture the remaining

trophectoderm fragments (Cencic and La Bonnardiere 2002). Regardless of culture method,

these cell lines share similar characteristics and are described as epithelial-like growing in a

monolayer of adherent cuboidal cells (Flechon, Laurie et al. 1995; Talbot, Caperna et al. 2000;

Shimada, Nakano et al. 2001; Cencic and La Bonnardiere 2002). Other common characteristics

were a prominent nucleus, and cytoplasmic structures resembling lipid-containing vesicles

(Flechon, Laurie et al. 1995; Talbot, Caperna et al. 2000; Cencic and La Bonnardiere 2002).

Upon reaching confluence, cells formed dome structures that would eventually form discrete

vesicles that floated in the medium (Talbot, Caperna et al. 2000; Shimada, Nakano et al. 2001).

These characteristics were common across species and in cell lines grown with or without feeder

layers.

A well characterized, feeder-dependent porcine trophoblast cell line, termed TE1, was

isolated from a 9 day preimplantation blastocyst (Flechon, Laurie et al. 1995). Morphologically,

these cells appeared to be a polarized epithelium that was positive for the pig trophectoderm

antibody, SN1 (Flechon, Laurie et al. 1995). A feeder-independent porcine trophoblast cell line

has been recently reported and, in addition to being positive for the SN1 antibody, was able to

functionally secrete interferon-γ into the culture medium (Cencic and La Bonnardiere 2002).

Recently, trophoblast stem cells, seemingly capable of forming only trophoblast

derivatives, have been isolated from the mouse (Tanaka, Kunath et al. 1998). Tanaka et al.

described these cells as “similar to the epithelial-type cells that appear during the isolation of ES

cells and to a trophectoderm cell line established from porcine blastocyst”. Critical requirements

for cell proliferation in trophoblast stem cell lines isolated by Tanaka et al. include FGF4 and a

growth medium conditioned by embryonic fibroblasts. Removal of FGF4 led to spontaneous

11

differentiation into trophoblast giant cells and the expression of a range of genes characteristic of

terminally differentiated trophoblast cells (Tanaka, Kunath et al. 1998). It is unclear whether any

of the previous trophoblast cell lines are truly stem cells. They have not been well studied with

regard to either gene expression or their capacity to differentiate in response to external cues

(Roberts, Ezashi et al. 2004).

Interferon-γ

As in ruminant species, the conceptus of the pig secretes proteins belonging to the

interferon family (Bazer 1989). Interferons are pleiotropic cytokines which, in addition to a

potent antiviral activity, exert multiple effects on cell growth and differentiation, specifically on

cells of the immune system (La Bonnardiere 1993). In pigs, two different interferons have been

shown to be developmentally induced and secreted by the trophectoderm cells of the implanting

embryo (Lefevre, Martinat-Botte et al. 1990; La Bonnardiere, Martinat-Botte et al. 1991). The

more abundant of these proteins is interferon-γ (IFN-γ) whose production culminates on Day 15

of development (La Bonnardiere 1993). To date, the function of IFN-γ is unknown.

The IFN-γ gene was first cloned and expressed from human peripheral blood leukocytes

(Gray, Leung et al. 1982). Subsequently, the structure and nucleotide sequence of porcine IFN-γ

was determined (Dijkmans, Vandenbroeck et al. 1990). Two types of interferons have been

described. Type I interferons contain α, β, and τ while Type II interferon include only γ (Cencic,

Henry et al. 2002). The Type I interferons have minimal structural homology with the Type II

interferon and there is evidence for only one IFN-γ gene in all mammalian species (Cencic,

Henry et al. 2002). At maturity, IFN-γ polypeptide has a mass of 14.74 kDa, corresponding to a

C-terminal cleavage of 17 residues from an expected 143 amino acid sequence (Cencic, Henry et

al. 2002). In comparison with human and mouse IFN-γ, the active form of porcine embryonic

12

IFN-γ is probably a homodimer under physiological conditions (Arakawa, Hsu et al. 1986;

Vandenbroeck, Dijkmans et al. 1991). There are no disulfide bonds present since the mature

IFN-γ is lacking any cysteine residues, thus the monomers are held together exclusively by non-

covalent forces (Cencic, Henry et al. 2002). The secondary protein structure is primarily alpha-

helical, with six helices in each subunit that comprise 62% of the structure (Ealick, Cook et al.

1991).

Interferon-γ secretion and synthesis by the implanting conceptus has been confirmed by

several methods (Lefevre, Martinat-Botte et al. 1990). Significant antiviral activity was found in

porcine uterine flushings and in conceptus-conditioned culture medium on Days 11-17 of

pregnancy (Cross and Roberts 1989; La Bonnardiere, Martinat-Botte et al. 1991). This antiviral

activity was confirmed by Mirando et. al as they found IFN-γ secretion by the pig conceptus

beginning on Day 12 of gestation, reaching a maximal level at day 15-16 and then declining in

production (Mirando, Harney et al. 1990; La Bonnardiere, Martinat-Botte et al. 1991).

Immunohistochemistry performed on thin sections of pig conceptuses showed that the cells of

the extraembryonic trophectoderm intensively secrete IFN-γ at their apical pole (La Bonnardiere

1993). Recently, it has been shown that polarized pig trophoblastic cell lines established from

Day 13-15 conceptuses can spontaneously secrete IFN-γ into the culture medium (Cencic and La

Bonnardiere 2002).

Unlike the ruminant species, the porcine trophoblastic interferons do not play an obvious

role in the maternal recognition of pregnancy (La Bonnardiere, Martinat-Botte et al. 1991). In

the pig, the possible roles of embryonic IFN-γ are only speculative. Researchers have shown that

the extraembryonic trophectoderm produces large quantities at a developmentally significant

time; around the time of implantation (Cross and Roberts 1989; La Bonnardiere, Martinat-Botte

13

et al. 1991). The porcine trophoblast cells do not possess any receptors for interferons at the

time of IFN-γ expression, ruling out an autocrine activity (Cencic, Henry et al. 2002). Studies

have demonstrated that porcine embryonic IFN-γ does not contribute to the maintenance of

corpus luteum functions (Lefevre, Martinat-Botte et al. 1998), but two possible functions of IFN-

γ have been proposed. The known biological roles of leucocytic IFN-γ suggests that

trophoblastic IFN-γ could act as an anti-infectious agent, providing a healthy environment for

implanting embryos (Cencic, Henry et al. 2002) or trophoblastic IFN-γ could remain confined to

the uterine lumen and be a direct effector of uterine epithelial depolarization via the apical side

of the luminal epithelium, leading to partial or profound remodeling (depolarization or other

effects) of this maternal tissue, a condition necessary for conceptus implantation (Cencic, Henry

et al. 2002).

Bone Morphogenetic Protein 4

Bone morphogenetic Protein 4 (BMP-4) is a member of the transforming growth factor

beta (TGFβ) superfamliy. Bone morphogenetic proteins were originally isolated by their ability

to induce ectopic bone and cartilage formation (Urist 1965). It is now known that this family of

proteins exerts a wide spectrum of biological responses on a large variety of cell types

(Balemans and Van Hul 2002). Many members of this family of proteins have important

functions during embryonic development in pattern formation and tissue specification. In adult

tissues they are involved in wound healing, bone repair, and bone remodeling (Balemans and

Van Hul 2002).

Bone morphogenetic proteins are synthesized as large precursor proteins comprised of a

signal peptide, prodomain, and mature domain (Ducy and Karsenty 2000; Shimasaki, Moore et

al. 2004). After a proteolytic cleavage of the signal peptide, the proproteins dimerize

14

(Shimasaki, Moore et al. 2004). A distinguishing structural feature of the TGFβ superfamily is

the presence of seven conserved cystine molecules which are involved in the formation of a

unique structure called a cystine knot (Vitt, Hsu et al. 2001). One conserved cystine residue is

not involved in the knot formation and makes a single disulfide bridge between the two subunits

that results in the dimerization essential for biological activity (Shimasaki, Moore et al. 2004).

The TGFβ signaling pathway is mediated by a family of serine/threonine receptor kinases

(Yamashita, Ten Dijke et al. 1996; Ducy and Karsenty 2000; Miyazono, ten Dijke et al. 2000;

Balemans and Van Hul 2002). These receptors fall into two classes: Type I and Type II. The

Type II receptor binds the ligand with high affinity, resulting in the recruitment of the Type I

receptor to form a ligand-receptor complex (Yamashita, Ten Dijke et al. 1996; Baker and

Harland 1997; Miyazono, ten Dijke et al. 2000). Upon dimerization, the Type II receptor

phosphorylates the Type I receptor in a cluster of glycine and serine (GS) residues near the cell

membrane, known as the GS domain (Figure 1). Phosphorylation of the GS domain stimulates

intracellular responses to the bound ligand (Wrana, Attisano et al. 1994; Yamashita, Ten Dijke et

al. 1996; Baker and Harland 1997; Miyazono, ten Dijke et al. 2000). Regulation of this pathway

is controlled by the function of cytoplasmic transduction molecules, the Smads (Baker and

Harland 1997). While many signaling pathways use a cascade of cytoplasmic molecules, the

Smads are the only cytoplasmic components known of in the TGFβ pathway (Baker and Harland

1997). Smad1, Smad5, and most likely Smad8 are substrates for BMP receptors and upon

phosphorylation, the receptor-specific Smads hetero-oligomerize with Smad4 and translocate to

the nucleus, where they regulate the expression of specific genes (Baker and Harland 1997; Hill,

Burghardt et al. 2000; Balemans and Van Hul 2002).

15

Figure 1 – Cascade of BMP signaling (Balemans and Van Hul 2002)

As previously stated, the first BMPs were isolated for their ability to induce ectopic bone

and cartilage formation in rodents (Urist 1965). However, studies have demonstrated a broader

range of biological activities in various cell types, including monocytes, epithelial cells,

mesenchymal cells, and neuronal cells (Balemans and Van Hul 2002). Bone morphogentic

protein 4 has been shown to play important roles in the establishment of the basic embryonic

body plan, in morphogenesis, apoptosis, and the development of organs and tissues (Hogan

1996). There is evidence for an inherent BMP system in the mouse and human placenta. In

mouse embryos, the expression of BMP-4 in the epiblast derived tissues has been implicated in

the development of the vascular connection between the placenta and the embryo (Fujiwara,

Dunn et al. 2001). In the mouse placenta, BMP-8b is expressed in the trophoblast in the

16

labyrinth region (Zhao, Liaw et al. 1998), BMP-4 is expressed in the spongiotrophoblast, and

BMP-7 is highly expressed in the trophoblast giant cells (Ozkaynak, Jin et al. 1997). As a result,

the hypothesis emerged that the expression of BMPs in the placenta may be involved in

trophoblast differentiation (Shimasaki, Moore et al. 2004). In humans, BMP-4 has been shown

to differentiate embryonic stem cells to trophoblastic lineages (Xu, Chen et al. 2002). It remains

unclear whether BMP-4 is the signal for trophoblast differentiation in vivo.

Summary

Proper development of the placenta is crucial for successful fetal development across all

the eutherian mammals. Placental abnormalities are well documented in SCNT pregnancies and

have been implicated as a major factor in SCNT pregnancy losses, contributing to the low

efficiency rate. However, these placental abnormalities have received less attention from

researchers in the field. A feeder-free porcine trophoblastic cell culture is thus deemed important

as a model for placental development could be used to address the molecular and cellular events

associated with normal and, in the case of cloned pregnancies, abnormal placental development.

The research that follows addresses this issue.

17

CHAPTER 3

CHARACTERIZATION OF PORCINE TROPHECTODERM DERIVED CELLS IN THE PRESENCE OF HBMP4 IN THE ABSENCE OF A FEEDER LAYER

Introduction

Numerous placental abnormalities have been associated with cloned progeny (Stice,

Strelchenko et al. 1996; Cibelli, Stice et al. 1998; Lewis, Peura et al. 1998; Hill, Roussel et al.

1999; Wells, Misica et al. 1999; Hill, Burghardt et al. 2000; Inoue, Kohda et al. 2002; Ogura,

Inoue et al. 2002). Malformed placentas are a contributing factor to the low cloning efficiency

reported across species, ranging from 0 – 7.2% (Wilmut and Peterson 2002). Reports involving

the production of cloned pigs do not mention the presence or absence of placental defects;

however, the absence of such observations should not discount the fact that they might occur. As

seen by the low cloning efficiency in pigs, ranging from 0.1 – 0.9% (Wilmut and Peterson 2002),

placental anomalies within the first trimester may be a contributing factor to this low efficiency.

This is supported by studies in which gestational losses were reported in cloned pigs between

days 21-40 (De Sousa, Dobrinsky et al. 2002; Lai, Park et al. 2002); temporally associated with a

significant event in placental development, implantation (Dantzer 1985). Although indirect, this

evidence suggests the high fetal and embryonic death that occurs during gestation of cloned pig

pregnancies can be attributed to malformed placentas.

The isolation of a trophoblast cell line with the use of a mouse embryonic fibroblast

(MEF) feeder layer has been described in several species (Ramsoondar, Christopherson et al.

1993; Flechon, Laurie et al. 1995; Tanaka, Kunath et al. 1998; Talbot, Caperna et al. 2000). It is

generally accepted that the MEFs provide an extracellular matrix to which the cells adhere for

survival and growth. It is also believed that soluble factors produced by the MEFs are required

18

for maintenance of these cells. Fibroblast growth factor 4 (FGF4) is one of these growth factors

(Tanaka, Kunath et al. 1998). Studies in mice have suggested that stem cells exist in the

extraembryonic ectoderm that forms from the polar trophectoderm (Tanaka, Kunath et al. 1998).

Trophoblast stem cells have been isolated from mouse blastocyst and continuously cultured

(Tanaka, Kunath et al. 1998). However, investigators have shown that bovine trophoblast cells

(Shimada, Nakano et al. 2001), and more recently porcine trophoblast cells can be propagated

without a feeder layer (Shimada, Nakano et al. 2001; Cencic and La Bonnardiere 2002).

There is a need to study both in vitro and in vivo development of the pig placenta, since

abnormal placental development has been implicated as a factor involved in somatic cell nuclear

transfer pregnancy losses (Willadsen, Janzen et al. 1991; Stice, Strelchenko et al. 1996; Cibelli,

Stice et al. 1998; Hill, Roussel et al. 1999; Wells, Misica et al. 1999; Hill, Burghardt et al. 2000;

Inoue, Kohda et al. 2002; Ogura, Inoue et al. 2002). A porcine trophoblast stem cell line can be

studied to enhance our understanding of the molecular and cellular events in normal and, in the

case of cloned pregnancies, abnormal placental development. The use of a feeder-free culture

system eliminates a potential source of contaminating cells that could confound biochemical and

molecular analysis; whereby excluding undefined elements affecting trophoblast cell

proliferation and differentiation and may in turn lead to a better understanding of the overall

effects growth factors have on trophoblast cell proliferation. The addition of the BMP4 growth

factor, part of the TGFβ signaling pathway, to human embryonic stem cells has been shown to

direct differentiation towards trophoblast lineages and play a role in the maintenance of

trophoblast cells in vitro (Xu, Chen et al. 2002). There are several inhibitors of BMP signaling

including Noggin, Chordin, Follistatin, Cerberus, Xnr3 and TSG. Conducting studies in vitro on

19

feeder-free trophoblast cell lines is an efficient method to investigate the role these factors play

in placental development.

The preimplantation embryo of the pig has been shown to secrete a series of proteins

belonging to the interferon family (Lefevre, Martinat-Botte et al. 1990; La Bonnardiere,

Martinat-Botte et al. 1991). The most abundant of these is interferon-γ (Lefevre, Martinat-Botte

et al. 1990; La Bonnardiere, Martinat-Botte et al. 1991). Interferon-γ is produced in a transient

manner by the trophectoderm of the porcine embryo between days 12 and 20 (Mirando, Harney

et al. 1990). Investigators have used interferon-γ expression as an identifying characteristic of

porcine trophoblast cells lines (Cencic and La Bonnardiere 2002). Alpha fetoprotein expression,

a known maker for endodermal lineages, has been used to distinguish trophoblast cell cultures

from endodermal cells (Xu, Chen et al. 2002) that line the trophectoderm at the time of embryo

collection.

The purpose of this study was to first determine if porcine trophoblast cell cultures from

in vivo produced embryos could be established in the absence of a feeder layer, and secondly if

medium supplemented with BMP4 enhances the proliferation and differentiation of porcine

trophoblast cells.

Materials and Methods

Primary cultures of trophoblast cells

Embryos were recovered from artificially inseminated prepuberal gilts. Superovulation

was induced with an intramuscular injection of 2000 IU of pregnant mare serum gonadotropin

(PMSG) followed by 750 IU of human chorionic gonadotropin (HCG) 72 hours later. The gilts

were artificially inseminated 24 and 48 hours after the HCG injection. Fifteen days post

conception, the gilts were hysterectomized and the reproductive tracts were immediately taken to

20

the lab. The conceptuses were flushed from the uterine lumen with 50 ml of Dulbecco’s

Phosphate Buffered Saline supplemented with 1% fetal calf serum and penicillin-streptomycin

(1X).

Elongated conceptuses were visualized under a dissecting scope and the embryonic disk

was located. The trophoblast was cut away from the embryonic disk (>3 mm margin) and

transferred into a 50 ml conical tube containing trophoblast cell medium [(TC) medium consist

of a 1:1 ratio of Dulbecco’s Modified Eagle Medium and Nutrient Mixture F-12 (DMEM/F12)

supplemented with 15% fetal calf serum, 0.1 mM 2-mercaptoethanol, 4 ng/ml FGF4 and 1X

penicillin-streptomycin]. The conical tube was placed in an incubator at 37°C and 5% CO2 until

further processing.

The tube was removed from the incubator and the contents were allowed to settle. The

supernatant was removed and the pellet placed in a fresh 50 ml tube in a volume of 15 ml of TC

medium. The trophoblast tissue was sheared into cell aggregates by drawing the tissue through a

16 gauge needle with a 20 ml syringe. This was centrifuged at 1000 x g for 5 minutes. The

supernatant was removed and the pellet was resuspended in TC medium and seeded in 35 mm

tissue culture plates coated with various cell substrates. Some trophoblast cell aggregates were

flash frozen in liquid nitrogen and stored at -20°C for gene expression assays.

Preparation of cellular substrates

Culture plates were coated with 1 ml of human extracellular matrix (HEM), collagen

Type IV, or Matrigel (all from Becton Dickinson, Bedford, MA). HEM was diluted to 10 µg/ml

in cold DMEM/F12. Collagen was diluted to 10 µg/ml in 0.05N acetic acid. Matrigel was

diluted 1:20 in cold DMEM/F12. Plates were incubated at 4ºC for at least overnight or at room

temperature for 1 hour. Before seeding cells, the plates were rinsed twice with TC media and

21

placed in the incubator at 37ºC and 5% CO2 to equilibrate. Mouse embryonic fibroblast feeder

layers (MEFs) were prepared as previously described (Abbondanzo, Gadi et al. 1993). Feeder

cells were thawed, mitotically inactived by treatment with mitomycin-C, and replated at 1.2x106

cells per 35 mm dish. MEFs were cultured for at least 2 days prior to the plating of trophoblast

cells.

Experiment 1 – Feeder-free cell culture

Trophoblast cell aggregates were randomly allocated to each treatment group (Figure 2).

The 9 treatment groups were HEM, collagen, or Matrigel coated plates in TC medium in the

presence or absence of human recombinant BMP4 (hBMP4) at 0, 10, or 20 ng/ml in factorial

design.

Cell aggregates were plated in 35 mm tissue grade culture plates, 3 plates per treatment

group prepared as described above in 500 µl of medium. Cultures were placed in the incubator

at 37°C and 5% CO2 and left undisturbed for 24 hours, after which time they were observed.

After cell attachment occurred, the medium volume was brought up to 2 ml and changed at 48

hour intervals. Cell growth was monitored by phase contrast microscopy and photographs taken

at 24 hours, 3 days and 7 days in culture.

After evaluation of the results Experiment 1, hBMP4 concentrations were adjusted to

evaluate a wider range and MEF feeder layers were added as an additional cellular matrix to

increase cell attachment and growth.

Experiment 2 – Feeder-free culture versus culture on a feeder layer

Trophoblast cell aggregates were randomly allocated to each treatment group (Figure 3).

The 12 treatment groups were HEM, collagen, or Matrigel coated plates in addition to mouse

22

embryonic feeder layers in TC medium in the presence or absence of hBMP4 at 0, 3, or 30

ng/ml.

Cell aggregates were plated in 35 mm tissue grade culture plates, 2 plates per treatment

group prepared as described above in 500 µl of medium. Cultures were placed in the incubator

at 37°C and 5% CO2 and left undisturbed for 24 hours, after which time they were observed.

After cell attachment occurred, the medium volume was brought up to 2 ml and changed at 48

hour intervals. Supernatant from each experimental group was collected at every media change

until the first passage and frozen at -20°C until further analysis. Cell growth was monitored by

phase contrast microscopy and photographs taken at 24 hours, 3 days and 7 days in culture.

Passaging

When primary cultures grew to confluence and colonies appeared with the

characteristics of previously described trophoblastic stem cell lines (Tanaka, Kunath et al. 1998),

those colonies were passaged. Secondary passage of the trophectoderm cell cultures was done

both enzymatically and manually. After the first enzymatic passage, this method was found to

be deleterious to the cells. The cells dissociated into a single cell suspension and only a small

proportion plated down. The cells that did plate down did not retain the trophoblast stem cell

morphology. Thus, all further passaging was done by manual passaging techniques only.

Cells were manually passaged using a pulled Pasteur pipette. The glass pipette was

prepared by heating it on a low flame and pulling the glass. The pipette was broken at the

desired diameter. The diameter of the pipette was determined by the size of the colonies,

approximately one third the size. The tip of the pulled pipette was then polished by passing it

gently over the flame. Using a dissecting scope inside a cell culture hood, colonies were

carefully cut using the edge of the pulled pipette into pieces of 10-100 cells. Once the colony

23

had been cut into the desired pieces, the harvested cells were transferred to a new 35 mm dish

with approximately 10 pieces in each dish. The dish was then returned to the incubator (37°C

and 5% CO2).

Gene expression assay

Total gene expression was measured using real time RT-PCR. Samples were taken from

the trophectoderm tissue used for the primary culture along with partial colonies from treatment

groups in the second experiment that were able to be further passaged. These samples were flash

frozen in liquid nitrogen and stored at -20°C until RNA isolation.

Total RNA was isolated from cells and tissue using the RNeasy Mini Kit (Qiagen)

followed by a treatment with RNase-Free DNase (Qiagen). DNase was removed from all

samples using the RNeasy MinElute Cleanup Kit (Qiagen) and stored at -20°C until analysis.

Reverse transcription was carried out using the High Capacity cDNA Archive Kit (Applied

Biosystems).

Real-time PCR amplification was carried out using the ABI Prism 7900HT Sequence

Detection System. Primer and probe sequences were designed by Applied Biosystems using the

Assay on Demand program based on the sequences of the porcine IFN-γ gene (Dijkmans,

Vandenbroeck et al. 1990) and the porcine alpha fetoprotein (AFP) gene (Kim, Nonneman et al.

2002). 18S rRNA was amplified as an endogenous control. Total RNA from whole

conceptuses, 15 days post conception, were used as a positive control to validate the

amplification efficiency of the primer probe sets. Standard curves were generated using a 10

fold dilution of control template ranging from 100 – 0.01ng per 25 µl reaction. All reactions

were preformed in a 96 well plate in triplicate. The slope of the standard curve was used to

estimate the efficiency of each gene amplification according to the formula:

24

efficiency = 10(-1/slope) – 1 (Pfaffl 2001).

The resulting efficiencies were: IFN-γ (95.6%), AFP (96.9%), and 18s (87.33%).

Real time RT-PCR was run on experimental samples in a 96 well plate in triplicate. As

negative controls, a reaction without template and a reaction without the reverse transcription

enzyme were preformed in triplicate for each IFN-γ, AFP and 18s in each 96 well plate. In

addition, the gene expression from the MEF feeder layer was included as a negative control.

Relative gene expression levels of IFN-γ and AFP were determined for each experimental

sample using ∆∆CT method normalized to 18s rRNA expression (Livak and Schmittgen 2001).

Antiviral Assay

Trophoblast cells were seeded onto 35 mm dishes as described above and overlaid with

2ml of TC medium. Every 48 hours, the medium was changed and supernatants were collected

from each experimental group in the second experiment and stored at -20°C. For interferon-γ

detection, the Porcine IFN-γ Colorimetric ELISA Kit (Pierce Biotechnology) was used according

to the manufacturer’s instructions.

ORO staining

To confirm that trophectoderm cells contained lipids in their cytoplasm, were stained

with oil red O. Cells from TSC4 were grown on chamber slides were washed with PBS, fixed in

3.7% paraformaldehyde for 2 minutes and then washed in water. An oil red O solution was

made by diluting 0.3% oil red O (Sigma) in 60% isopropanol and 40% water. The fixed cells

were incubated in the oil red O solution for 1 hour at room temperature. The oil red O solution

was aspirated and the dishes were washed in triplicate with water. Phase contrast microscopic

pictures were taken immediately following the staining.

25

Results

Cell Culture

Embryos were collected four times for Experiment 1. The first collection was from two

gilts with a superovulatory response. The resulting embryos were too numerous to count. Cells

were plated as described in Experiment 1 above in the Methods Section and termed TSC1.

Within 24 hours of culture, many trophoblast cell aggregates appeared to undergo necrosis,

however outgrowths of trophoblast cells appeared from the necrotic masses and grew outward in

a radial fashion. After three days of culture, many cell types were present. These cultures were

dominated by large cells with varying morphologies. Among these large cells were colonies of

smaller cells with epithelial type morphology that had a prominent nucleus and a high nuclear to

cytoplasmic ratio. The epithelial type cells grew in tight colonies with definite borders and

contained cytoplasmic structures resembling lipid-containing vesicles similar to trophoblast stem

cell morphology previously described in the literature (Tanaka, Kunath et al. 1998). These

colonies initially appeared on all matrices across all hBMP4 concentrations.

After seven days in culture the colonies developed distinct differences across groups

(Figure 4). Cell growth on collagen was pronounced with tight colonies having definite borders

among large cells. Colonies on collagen were larger and more pronounced in both the hBMP4

supplemented groups than when cultured without hBMP4. The Matrigel coated plates contained

large sheets of epithelial type cell growth instead of compact colonies. This type of growth

characteristic was present in all hBMP4 treatments on Matrigel. In contrast, few cells survived

and propagated on human extracellular matrix. Only small colonies having the trophoblast stem

cell morphology (Tanaka, Kunath et al. 1998) were among the large cells on HEM when cultured

26

in medium containing 10 ng/ml hBMP4. Cells were passaged and only cells growing on

Matrigel could be further cultured.

Embryos were collected a second (TSC2) and third (TSC3) time for Experiment 1 with

unsuccessful results. During the second embryo collection, only one gilt had a superovulatory

response. In the primary culture of TSC2, very little tissue attached and there was no cell

growth. The primary culture of TSC3 resulted from two gilts, however became contaminated

and was discarded.

The fourth and final embryo collection for Experiment 1 (TSC4) was from 2 gilts. The

results were similar to TSC1. Only cells growing on Matrigel were able to be further cultured.

By the third passage, only cells growing without hBMP4 supplementation contained cell

colonies similar to trophoblast stem cells (Tanaka, Kunath et al. 1998).

Embryos were only collected once for Experiment 2 (TSC5). Two gilts produced the

embryos that were used in the primary culture. Unlike the previous experiment, there was very

little tissue attachment across all matrices, except on MEFs. Trophectoderm tissue readily

attached to the feeder; however no cellular outgrowths were visible. After 7 days in culture,

there was very little cell growth across groups and no colonies with trophoblast stem cell

morphology (Tanaka, Kunath et al. 1998) previously described (Figure 5). Cell growth on

collagen contained various cell types including large vacuolated cells inter mixed with sheets of

epithelial cell growth. On HEM, the trophectoderm tissue began to dissociate from the plate and

few unidentifiable cells remained, but did not proliferate. Cell growth on Matrigel was similar to

that on HEM. There were no cellular outgrowths from the tissue attached on the feeders. These

characteristics were similar across all hBMP4 concentrations.

27

After 24 days in culture, no cells were growing on Collagen, HEM, or Matrigel that

possessed the desired morphology (Tanaka, Kunath et al. 1998). Cell growth on MEFs was

abundant, among the cell growth were colonies previously described in TSC1. These cell

colonies grew on top of the feeder layers with the central most cells tightly packed and filled

with lipid-like vesicles in their cytoplasm. The centers of these colonies were manually passaged

onto fresh feeders. Differences between hBMP4 concentrations began to appear at the second

passage (Figure 6). Cells growing in medium supplemented with 0 ng/ml or 3 ng/ml hBMP4

remained in tight compact colonies with small cells containing lipid in their cytoplasm. Several

cell colonies in the 30 ng/ml hBMP4 group spread out, proliferation slowed down and cells lost

the characteristic lipid droplets in their cytoplasm. In addition, these cells became large in size

almost transparent with the feeder layer visible through the colony. By the fourth passage, all

colonies supplemented with 30 ng/ml hBMP4 had lost the trophoblast stem cell morphology.

Only 2 colonies from each the 0 ng/ml and the 3 ng/ml hBMP4 concentrations could be

passaged. After the fourth passage, all colonies lost the desired morphology (Tanaka, Kunath et

al. 1998) and were unable to be further cultured.

Gene Expression

Interferon-γ expression was detected in three of the ten samples tested (Figure 7). Those

samples included the trophectoderm tissue from which the cultures were grown cultures as well

as the groups supplemented with 0 ng/ml and 30 ng/ml hBMP4 at the first passage. At the fourth

passage, there was no detectable IFN-γ expression in any hBMP4 concentration. There was also

no detectable IFN-γ expression by the MEF or the negative controls. When the expression levels

were analyzed using the ∆∆CT method (Livak and Schmittgen 2001), IFN-γ expression

decreased from the trophectoderm tissue by 707.9 fold in colonies supplemented with 0 ng/ml

28

hBMP4 and by 73.4 fold in colonies supplemented with 30 ng/ml hBMP4 (P<0.05). Alpha-

fetoprotein expression was only detected in the trophectoderm tissue used to grow these cultures

(Figure 8). There was no detectable AFP expression by the MEFs or the negative controls.

Oil Red O

Oil Red O staining confirmed that all experimental groups of TSC4 do contain lipid

droplets in their cytoplasm (Figure 9).

IFN-γ ELISA

Supernatant samples from TSC5, days 2-14 in culture, were positive for the presence of

IFN-γ in the culture medium. Quantitative analysis was not possible due to the large

concentration of IFN-γ in the samples; however the colorimetric response indicated a positive

result for the existence of IFN-γ.

29

Figure 2 – Feeder-free cell culture

30

Figure 3 – Feeder-free culture versus culture on a feeder layer

31

Figure 4 – Phase microscopic images of TSC1 after 7 days in culture.

32

Figu

re 5

– P

hase

mic

rosc

opic

imag

es o

f TSC

5 af

ter 7

day

s in

cultu

re.

Not

e th

e la

ck o

f cel

l gro

wth

.

33

Figure 6 – Phase contrast microscopic images of TSC5 after the first passage.

34

Figure 7 – Interferon-γ gene expression, TSC5. A – Amplification plot, B – Relative gene expression (Livak and Schmittgen 2001).

35

Figure 8 – α-fetoprotein expression, TSC5. A – Amplification plot, B – Relative gene expression (Livak and Schmittgen 2001).

36

Figure 9 – Phase contrast microscopic image of Oil Red O staining in trophoblastic cells.

37

Discussion

In this study, porcine trophoblastic cell cultures were initiated on HEM, collagen Type

IV, Matrigel and MEF feeder layers to determine if a trophoblast cell line from in vivo produced

embryos could be established in the absence of a feeder layer, and secondly if medium

supplemented with hBMP4 enhances the proliferation and differentiation of porcine trophoblast

cells.

The cultures of TSC1, TSC4, and TSC5 shared the same morphological characteristics of

earlier described trophectoderm cell lines. These cells grew in a monolayer of adherent cuboidal

cells that were epithelial-like in morphology (Flechon, Laurie et al. 1995; Talbot, Caperna et al.

2000; Shimada, Nakano et al. 2001; La Bonnardiere, Flechon et al. 2002). Similar to the cultures

described in the pig (Flechon, Laurie et al. 1995) and in the bovine (Talbot, Caperna et al. 2000),

these cells displayed a prominent nucleus and numerous lipid droplets in their cytoplasm as

confirmed by oil red O staining. In addition, primary cultures of TSC1, TSC4, and TSC5 were

morphologically similar to a trophoblast stem cell line described in the mouse (Tanaka, Kunath

et al. 1998), forming tight colonies with an epithelial morphology. Whether the trophoblast

cultures in this study or prior trophoblastic cell lines, with the exception of the mouse (Tanaka,

Kunath et al. 1998), described in the literature are in fact stem cells has yet to be determined.

Previous trophoblastic cell lines have not been studied well enough with regard to either gene

expression or cellular markers indicative of pluripotency. Lack of porcine sequence information

and porcine specific stem cell markers has hindered the characterization of the primary

trophoblast cultures in this study as trophoblast stem cells or trophoblast progenitor cells, one

step along the committed lineage.

38

In addition to the morphological similarities to previously described trophoblast cell

lines, a specific function of in vivo trophectoderm cells, IFN-γ expression, was maintained by

two groups of TSC5 up to the first passage. Real time RT-PCR detected IFN-γ expression in the

trophectoderm tissue used to begin this culture as well as in colonies collected at passage one

from groups growing on MEF feeders supplemented with 0 ng/ml or 30 ng/ml hBMP4 (Figure

7). This result is in accordance with the results of the IFN-γ ELISA. The antiviral assay showed

that all groups in the primary cultures of TSC5 secreted IFN-γ into the culture media up to 14

days in culture. This result is comparable to another porcine trophoblast cell line (La

Bonnardiere, Flechon et al. 2002). The functional characteristics of TSC5, in addition to absence

of AFP expression (Figure 8), ruling out an endodermal origin, demonstrate that these are in fact

trophoblast cells or trophoblast derivatives.

While cells did share morphological similarities, their attachment and growth varied

across different extracellular matrices. Matrigel contains mostly laminin, collagen IV, and

heparin sulfate proteoglycan derived from a mouse tumor cell line (Kleinman, McGarvey et al.

1982) and has been shown to support the undifferentiated growth of human embryonic stem cells

(Xu, Inokuma et al. 2001). Multiple passages on Matrigel were possible with TSC1 and TSC4;

however TSC5 would not grow on any matrix except the MEF feeder layer. This could be an

isolated incident and further biological replicates of the second experiment are necessary. While

Matrigel and MEF feeders could initiate cell growth, these cultures could not be maintained long

term regardless of hBMP4 concentration in the culture medium. Previous trophoblast cell lines

established and maintained in a feeder-free system have required a MEF conditioned medium to

proliferate (Shimada, Nakano et al. 2001; La Bonnardiere, Flechon et al. 2002). Knowing that

the MEF feeders secrete multiple factors into the culture media and that prior trophoblast cell

39

lines have not been established in the absence of either a MEF feeder layer or MEF conditioned

medium, it is plausible that factors other than FGF4 are required to maintain trophoblast cells in

a proliferative state. This study used a defined medium supplemented with FGF4, a growth

factor necessary to mouse trophoblast stem cells (Tanaka, Kunath et al. 1998), to examine the

effects of hBMP4 without the confounding effects of unknown growth factors produced by the

MEF feeders. Because a defined culture medium is important, until the unknown biological

factors in MEF conditioned medium are delineated, the clear effects of growth factors cannot be

tested in a feeder-free culture system.

In this study, though hBMP4 did not have an effect on the initial trophoblast outgrowths,

it did have an effect over time. The addition of hBMP4 to the porcine trophectoderm cultures

resulted in a progressive change in morphology and a decline in proliferation that was dose

dependent. While colonies in all groups eventually ceased to proliferate, the higher

concentrations of hBMP4 in the culture medium caused a change in trophoblast cell morphology

in as early as the first and second passages. Large vacuolated cells with less lipid appeared on

the edge of colonies and ultimately, the entire colony lost the tight epithelial morphology that is

characteristic of trophoblast stem cells (Tanaka, Kunath et al. 1998). If the initial cultures in this

study were in fact trophoblast stem cells or trophoblast progenitor cells, this change in

morphology would be indicative of differentiation into trophoblastic derivatives. Similar to a

mouse trophoblast stem cell line (Tanaka, Kunath et al. 1998), upon differentiation large cells

developed and ceased to grow.

This is supported by the IFN-γ expression pattern. In vivo, IFN-γ production by the

trophoblast cells peaks at day 16 and then slowly declines until day 20 (Cross and Roberts 1989;

La Bonnardiere, Flechon et al. 2002). Since embryos were collected 15 days post conception,

40

nearing the peak of IFN-γ production, it is not surprising that IFN-γ expression was high in the

trophectoderm tissue used to establish these cultures (Figure 7). The decline in expression at

passage one and the eventual disappearance of IFN-γ expression by passage four could represent

the differentiation of these cultures similar to the drop off in IFN-γ production associated with

developmental progression past day 20 in vivo. Comparable to work done in human trophoblast

differentiation (Xu, Chen et al. 2002), these data suggest that hBMP4 potentially plays a role as a

trophoblast differentiation cue in vitro.

Conclusion

This study has shown that the cell type isolated is trophoblastic in origin and both cell

substrate and hBMP4 affect initial trophoblast outgrowths. These data lead us to propose that

addition of hBMP4 to the culture medium directs the differentiation of trophoblast progenitor

cells into trophoblast subtypes. Recent studies on the effects of hBMP4 on human embryonic

stem cells have reached the same conclusion (Xu, Chen et al. 2002). While BMP4 is a good

candidate as the signal for trophoblast differentiation in vitro, further research in necessary to

determine whether BMP4 is the signal for trophoblast differentiation in vivo.

In addition, the results of this study have shown that cultures of porcine trophectoderm

tissue collected 15 days post conception can not establish cell lines with long term proliferative

capacity either on MEF feeder layers or on an extracellular matrix. This is perhaps due to the

absence of unknown biological factors in MEF conditioned medium, extracellular cues from

exposure to cellular matrices, the age at which the embryos were collected, or a combination of

these issues.

This research should be continued as the potential applications are many. Since it is

problematic to manipulate the postimplantation embryo to study placental development, the

41

continuous culture of porcine trophectoderm cells may provide a good in vitro model. Once a

model is established, developmental studies can be performed to supplement our limited

knowledge of the morphological, cellular and molecular processes in the extraembryonic

membranes. The lack of information about the placenta limits our understanding of critical

events in pregnancy. Insight into normal placental development would be valuable since

abnormal placentas are common in SCNT pregnancies and might contribute to early embryonic

death.

If this research should continue, several things should be taken into consideration in

future studies. Trophoblast cells require either a MEF feeder layer or MEF conditioned medium

to remain in a proliferate state; however, a defined feeder-free system is necessary to simplify the

study of supplemented growth factors added to the culture medium. For those reasons, MEF

conditioned medium should be critically evaluated to delineate the biological factors, other than

FGF4, that are essential to trophoblast cells. Once there is an optimized and defined medium

that can support long term trophoblast cell growth on an extracellular matrix such as Matrigel,

then studies can be continued with out the reliance on MEF feeder layers.

Furthermore, younger embryos might be a better source for the isolation of trophoblast

stem cells in the pig. Previous studies in the mouse first attempted to isolate trophoblast stem

cells from an embryo 6.5 days post conception (dpc) without success (Tanaka, Kunath et al.

1998). By using an earlier embryo, 3.5 dpc, they were able to successfully isolate a trophoblast

stem cell line, termed TSC3.5 (Tanaka, Kunath et al. 1998). At 3.5 dpc, the mouse embryo has

not yet implanted into the uterine epithelium and is in between Theiler stages 3 and 4 (Burger,

Davidson et al. 2004). During this intermediate stage in development, the mouse embryo is a

spherical blastocyst that is still contained in, or is just beginning to hatch from the zona pellucida

42

(Burger, Davidson et al. 2004). This is equivalent to a porcine embryo 6 dpc (Patten 1948), and

it would be useful to try and isolate trophoblast stem cells from a porcine embryo at this early

stage. Additionally, pig embryos 6 dpc have not yet undergone elongation (Patten 1948) and

could be cultured individually in order to maintain a genetic identity for each cell line.

A question to address in future trophoblast research is why imprinting information is

disrupted in SCNT embryos. Aberrant patterns of DNA methylation and imprinted gene

expression have been reported for cloned embryos in different species (Bourc'his, Le Bourhis et

al. 2001; Kang, Koo et al. 2001; Ohgane, Wakayama et al. 2001; Inoue, Kohda et al. 2002;

Ogura, Inoue et al. 2002) and in some cases it appears that the extra-embryonic tissues are

particularly affected (Inoue, Kohda et al. 2002; Ogura, Inoue et al. 2002). Once a trophoblast

model is established from a conventional pregnancy, the function of those imprinted genes and

the pathways they play a role in can be described which would provide insight into why the

disruption in their gene expression may result in the physical anomalies associated with the

cloned phenotype.

Since the early embryonic loss seen in porcine SCNT pregnancies is temporally

associated with implantation, it would be interesting to look the factors secreted by the

trophectoderm during this important time frame. It is possible that a disruption in the production

of factors normally secreted by the trophectoderm of the implanting conceptus may interfere

with the cellular remodeling and subsequent interaction between the trophoblast and the uterine

epithelium that is necessary for implantation and the establishment of pregnancy. Additionally,

estrogen related genes should be studied, as an anomaly in their expression pattern could result in

the failure of maternal recognition or pregnancy. Without proper estrogen secretion by the

implanting conceptus, prostaglandin-F2-alpha secretion does not transition from an endocrine

43

direction to an exocrine direction, the corpus luteum lyses, and pregnancy does not proceed

(Bazer 1989).

Once culture conditions are optimized, it would be interesting to flush embryos from

conventionally bred gilts in addition to flushing SCNT and parthenogenetic embryos as a

potential third experiment. These embryos would be collected at various stages. Embryos 6 dpc

would be useful for trophoblast stem cell isolation. In addition, collecting embryos at 15, 20 and

25 dpc would give insight into the cellular and molecular events occurring before, during, and

immediately after implantation. Cells isolated from the trophectoderm of each group could be

compared for differences in morphology as well as gene expression. DNA and protein content of

the trophectoderm could be used to look for hyperplasia and hypertrophy associated with SCNT

placentas. In addition, aromatase activity in these cells would provide insight into the possibility

that the early embryonic losses seen in SCNT pregnancies result from a lack of estrogen, the

signal for maternal recognition of pregnancy.

The availability of trophoblast stem cell lines, which can be differentiated into

trophoblast subtypes in vitro, would open new possibilities for determining the function of genes

and signaling pathways that are important in normal placental development leading to a better

understanding for how abnormalities in these pathways might contribute to the early embryonic

death and the physical anomalies associated with SCNT.

44

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