Download Characterization of embryonic stem cells: A special focus on farm

Indian Journal of Biotechnology
Vol 8, January 2009, pp 23-32
Characterization of embryonic stem cells: A special focus on farm animals
D Kumar*, T Anand, M K Singh, M S Chauhan and R S Manik
Animal Biotechnology Center, National Dairy Research Institute, Karnal 132 001, India
Received 12 September 2007; revised 26 May 2008; accepted 17 July 2008
Embryonic stem (ES) cells are derived from the inner cell mass of blastocysts. They grow indefinitely while maintaining
the pluripotency in the presence of specific growth factors such as leukemia inhibitory factor. The molecular mechanisms
for self-renewal of pluripotent cells and the role of various growth factors involved in self-renewal as well as differentiation
are being deciphered. ES cells, in their undifferentiated state, are characterized by a distinct morphology and by the presence
of a set of markers classified into intracellular and extracellular types. Expression of specific markers (surface and
transcription based) is an important criterion for pluripotent or undifferentiated state of cells. The expression of these
markers is found to be exclusive to a particular species. A thorough understanding of the expression of these markers and of
factors or conditions for the long-term culture of ES cells, without compromising their pluripotency and a stable genetic
make-up is very important for the production and maintenance of ES cells from different species of farm animals. In this
review, we present an overview of characterization of embryonic stem cells in farm animals.
Keywords: Embryonic stem cells, characterization, differentiation, karyotyping, epigenetics, farm animals
Introduction
Research on stem cell is advancing the knowledge
about how an organism develops from single cell and
how healthy cells replace damaged cells in adult
organism. There are two kinds of stem cells:
embryonic stem (ES) cells and adult stem cells. A
blastocyst consists of an outer layer of cells called
trophectoderm (TE) and an inner mass of cells called
inner cell mass (ICM). ES cells are isolated from the
ICM of the blastocyst. These cells are pluripotent in
nature i.e. they are able to produce any kind of cells or
tissues other than extra embryonic cells. Thus, ES
cells are undifferentiated, unspecialized cells that can
renew themselves for long periods through cell
division and also give rise to one or more specialized
cell types with specific functions in the body.
Specialized cells are those, which are committed to
conduct a specific function, but ES cells remain
uncommitted, until they receive a signal for
differentiation into specialized cells. To maintain
undifferentiated state, ES cells require a substrate
such as feeder cells or matrix and specialized media
components. The work in this area was initiated by
Evans and Kaufman1, who discovered that permanent
pluripotent cell lines could be derived directly from
the ICM of blastocysts of mouse. These ES cells were
originally derived and maintained on feeder layers.
However, now it has been found that they could also
be maintained in feeder free culture conditions in
which the media are supplemented with leukemia
inhibitory factor (LIF). Activation of the LIF pathway
appears to be required for self-renewal of mouse ES
cells. The human cells do not have the same response
to LIF, suggesting that mouse ES cells and hES cells
require different signals to maintain self-renewal and
pluripontency. The farm animal ES-like cells are also
normally cultured on feeder cells because the
molecular pathways and key molecules required for
maintaining pluripotency in these species are
unknown2.
Attempts have been made in a number of
mammalian species to establish embryo-derived cell
lines. Pluripotent cell lines of human ES cells have
also been established by several groups3,4. In addition
to mice and human, attempts have also been made to
establish ES cell lines from other mammals like rat5,
pig6, mink7, bovine8, equine9, sheep10, rabbit11 and
rhesus monkey12. Therefore, identification of reliable
markers for the characterization of ES cells is of great
importance in order to exploit their potential.
____________
*Author for correspondence:
Tel: 91-184-2259301; Fax: 91-184-2250042
E-mail: [email protected]
Characterization of ES cells
There are some important criteria which needs to
be satisfied before a stem cell line qualify to be a
24
INDIAN J BIOTECHNOL, JANUARY 2009
bonafide ES cell line. These include morphological
similarities to ES cells of other species, indefinite
undifferentiated proliferation in vitro, potential to
differentiate into three embryonic germ (EG) layers
and expression of specific molecular markers and
maintenance of normal karyotype. It is essential to
test generated ES cell line, whether they have
fundamental properties to make them as ES cells, the
process is called characterization. Numerous methods
have been reported for the characterization of ES cell
lines from mammals. Studies in mice1,13-17 have used
cell morphology, biochemical markers, expression of
surface markers, transcription factor expression,
ability to differentiate into various cell types and
tissue types and participation in embryonic
development as the criteria for validating the stem cell
nature of ES cell lines. In addition, extended
proliferative capacity and pluripotency while
maintaining a normal karyotype are important
characteristics of these cells. Recent evidence
suggests that the epigenetic status of the cells is
also an important criterion for characterization
of ES cells18.
Morphology of ES cell colonies
Mouse and human ES cell colonies have a
characteristic morphology. They usually proliferate in
tight round shaped colonies with smooth edges1. The
morphology of ES cells has two important traits–they
have quite small amount of cytoplasm and they
exhibit faster proliferation rate in a given population
of cells. However, for many animals such as cow,
sheep, pig, horse, hamster, mink, rabbit, and primate,
embryo derived are found to propagate as flattened
colonies, almost as a monolayer with individually
distinct cells that have been described as epitheliallike or epitheloid19, whereas in case of buffalo the
primary colonies have been observed to be dome
shaped with abundant lipid-like vacuoles20.
Cytogenetic Analysis
The karyotyping of ES cells has recently received
much attention in most laboratories; this has been
studied by G-banding, with most ES cells exhibiting a
normal compliment of chromosomes. There are now
several reports regarding assessment of karyotype in
long-term cultures. Analysis of ES cells lines, for
example, reveals normal karyotype at passage
numbers ranging from 24-14021. Karyotyping is
generally performed at different passages in order to
know the genetic stability of ES cells during culture
and in this respect various groups have found normal
karyotyping in ES cells of different species such as
buffalo20, bovine22 and sheep23.
Expression of Various Surface Markers
Alkaline Phosphatase
Alkaline phosphatase (ALP) enzyme is secreted by
almost all the cells but it’s intensity is found to be
higher in undifferentiated cells. ALPs located at the
cell surface are linked to the cell membrane via a
phosphatidylinositol glycan linkage. Although they
are classified on the basis of their ability to hydrolyse
orthophosphate monoesters at alkaline pH, their role
under more physiological conditions remains largely
unknown. The ES cells are known to express a high
specific activity of ALP, which declines during
progressive differentiation resulting in low ALP
activities in differentiated cells. Mouse ES cells
display ALP activity24,25, as do most tested human ES
cell lines26-28 but the signal for this pluripotent cell
marker seems to be variable in bovine ES cell-like
cells8,29-33. ALP has been used as a marker for porcine
ES cells34,35, sheep ES cell lines23,36, canine stem cell
like cells37 and buffalo ES cell-like cells38,39 and by
many groups in bovine40-42 as an indicator for ES
cells.
Stage Specific Embryonic Antigens (SSEAs)
The accessibility of molecules on the surface of
cells make them exceptionally convenient markers for
characterizing cell types, often recognized as antigen
by specific antibodies. Cell surface antigens provide
an invaluable tool for analyzing and sorting cells that
have particular characteristics within specific
contexts. A number of surface markers have been
used for the characterization of ES cells (Table 1).
SSEAs, which include SSEA-1, SSEA-3 and SSEA-4,
are developmentally regulated during early
embryogenesis and are widely used as markers to
monitor pluripotency of ES cells. These SSEAs are
various types of glycoproteins or glycolipids. SSEA-1
antigen, which is associated with α 1-3 fucosylated
N-acetyl–lactosamine43, first appears at 8-cell stage
mouse embryo44. Mouse primordial germ cells
express high levels of SSEA-1 and their colonies
cultured in vitro have strong cell-cell adhesion45,46.
The presence of SSEA-1 has not been documented in
human embryos47, however, pig, canine, chicken and
sheep ESCs express SSEA-13,19,23,37,48. Conflicting
reports are available in bovine on the presence of
SSEA-1 in ES cells, with some authors reporting its
KUMAR et al.: CHARACTERIZATION OF EMBRYONIC STEM CELLS
25
Table 1Surface markers expressed by pluripotent cells in different species
Marker
Bovine
Buffalo
Murine
Human
Alkaline phosphatase
+
+
+
variable
SSEA-1
Variable
??
+
-
Bovine-50
Mouse and humans-27
SSEA-3
Variable
Variable
-
+
Bovine-49, 50
Buffalo-38, 57
Mouse and human-27, 59
SSEA-4
Variable
+
-
+
Bovine-41, 49, 50
Buffalo-38, 57
Human-59
TRA-1-60
-
Variable
-
+
Mouse-47, 60
Buffalo-38, 57
Human-3, 47, 53, 59, 61
TRA-1-81
-
+
-
+
Mouse-47, 60
Buffalo-38, 57
Human-3, 9, 47, 53, 58, 61
TRA-2-49
??
-
+
Human-47, 53, 56
TRA-2-54
??
-
+
Human-47, 53, 56
presence in ES cell like cells49,50 but others not finding
its expression41. Our group has not observed SSEA-1
expression in buffalo ES cells (unpublished data).
The other two most widely studied ES cell
pluripotency surface markers are SSEA-3 and
SSEA-4. They are related to globoseries cell surface
glycolipids that were first used to delineate
embryological changes in the developing mouse
embryo51,52. Both of these antigens were found to
recognize a sequential region of mouse ganglioside
epitope, with SSEA-4 recognizing the terminal
portion of sequenence and SSEA-3 recognizing the
internal region of sequence. Surface markers like
SSEA-3 and SSEA-4 were originally identified on
human EC or ES cells12,27,47,53,54. Also, ES cells from
primates like rhesus monkey show expression of
SSEA-3 and SSEA-412. In mouse ES cells, SSEA-3
and SSEA-4 are expressed in 2 to 8-cell and morula
stages of preimplantation embryos and are also found
on unfertilized oocytes; however, there is a loss of
expression in the ICM of mouse embryos51,52. In
contrast, in human embryos, there is no expression of
SSEA-3 or SSEA-4 at 2 to 8-cell or morula stages;
however, these are expressed on the ICM of human
blastocysts and on isolated human ES cells47. It has
been well documented that the expression of these
cell surface markers changes both with development
References
Bovine-29, 33
Buffalo-20, 38, 39
Human-26-28
Mouse-25
and differentiation in vitro55,56. Whereas SSEA-3 and
SSEA-4 are expressed in sheep23, attempts towards
the detection of these markers in cattle ES cell
colonies have been found to give variable results33.
Immunohistochemical staining of bovine ES cell like
cells has demonstrated the expression of SSEA-3 and
SSEA-441,49,50. The buffalo ES cell like cells also
showed the expression of SSEA-338 and SSEA-438,57.
However, our group has observed lack of expression
of SSEA-3 in buffalo ES cells (unpublished data).
Tumour Rejection Antigens
Another class of surface markers for pluripotent
cells is that of tumour rejection antigens (TRA) series.
TRA-1-60 reacts with a sialidase-sensitive epitope
while TRA-1-81 reacts with an unknown epitope of
the same molecule. TRA-1-60 and TRA-1-81 were
originally identified on human EC and ES
cells12,27,47,53,54,58. ES cells from primates like rhesus
monkey have also been shown to express TRA-1-60
and TRA-1-812. The expression of TRA-1-6038 and
TRA-1-81 has also been reported in buffalo ES cell
like cells38,57.
Expression of Transcription Based Markers
A number of transcription factors that play critical
role in maintaining stem cell self-renewal have been
26
INDIAN J BIOTECHNOL, JANUARY 2009
identified, and analysis of their expression is being
used to characterize ES cells in different species
(Table 2). Oct4, Sox2, Nanog, Foxd3 and Rex1 are
thought to be central to the transcriptional regulatory
hierarchy that specifies ES cell identity because of
their unique expression patterns and essential role
during early development62-68. It is likely that these
key stem cell regulators bind and regulate genes
encoding other transcriptional regulators, which in
turn determine the developmental potential of these
cells, but knowledge of the regulatory circuitry of ES
cells and other vertebrate cells is lacking.
Oct4 is a transcription factor belonging to the class
V of POU family factors. The POU family of
transcription factors binds to the octamer motif ATGC
(A/T) AAT found in the regulatory domain of cell
type-specific as well as ubiquitous genes. The POU
domain comprises two structurally independent subdomains – the POU specific domain (POUS) and the
homeodomain (POUH) connected by a flexible linker
of variable length. The POU domain binds to the
DNA via interaction of the third recognition helix of
the POUH with bases in the DNA major groove at the
3’ A/TTA rich portion of octamer site. The POUS
domain exhibits site specific, high affinity DNA
binding and bending capability. Both the POUH and
POUS subdomains function as structurally
independent units with cooperative high affinity DNA
binding specificity. Functional cooperation between
the two subdomains may occur indirectly via DNA by
overlapping base contacts from the two subdomains.
In ES cells, Oct4 activates gene transcription
regardless of the octamer motif distance from the
transcriptional initiation site. The Oct4 is expressed
by all pluripotent cells during embryogenesis, and is
also abundantly expressed by undifferentiated mouse
ES cells69-71, as well as EG cell lines72. Oct4 deficient
mouse embryos only develop to a stage that looks like
a blastocyst, and although cells are allocated to the
interior, these blastocysts are actually only composed
of TE cells62. Oct4 has also been established as a
marker for human pluripotent ES cells. However, in
bovine cells, the usefulness of Oct4 has been
questioned by the identification of a bovine Oct4
pseudogene73. On the contrary, the bovine blastocysts
and primary cultures from ICM are found to express
exclusively the Oct4 ortholog and expression of the
pseudogene could only be detected in adult tissues
such as liver, kidney, and white blood cells by Yadav
and coworkers42. Recent data suggests that the
Table 2Transcription based markers of pluripotent ES cells
Marker Bov- Buff- Mur- Huine alo ine man
Reference
Oct4
+
+
+
+
Bovine-42, 50, 70, 73, 80, 81
Buffalo-20
Canine-37
Goat-74
Porcine-82
Nanog
??
??
+
+
Mouses and human-65, 66
Goat-74
Porcine-82
Sox2
??
??
+
+
Mouses and human-64, 81
Porcine-82
Foxd3
??
??
+
-
Mouses and human-83, 84
Rex1
??
??
+
+
Mouses and human-85-87\
Oct4-protein becomes restricted to the embryonic disc
of hatched bovine embryos33. The expression of Oct4
in undifferentiated pluripotent cells has also been
shown in various other species like canine37, goat74
and buffalo20.
Nanog is a homeobox-containing transcription
factor with an essential function in maintaining the
pluripotency of the ICM cells65. Furthermore, over
expression of nanog is capable of maintaining the
pluripotency and self-renewal characteristics of ES
cells under the conditions where normally the cells
would be exposed to differentiation-inducing culture
conditions66. Nanog transcripts appearing first in the
inner cells of the morula prior to blastocyst
formation65,66 are restricted to the ICM in the
blastocyst75 and are no longer detectable at
implantation. Expression of nanog reappears in the
proximal epiblast at embryonic day 6 in mouse and
remains restricted to the epiblast as development
progresses67. Nanog seems to be one out of the several
factors that are expressed in pluripotent cells and are
down regulated at the onset of differentiation. Nanog
mRNA was detected in the ICM but not in the TE of
expanded goat blastocysts74; a pattern that follows the
expression observed in mice.
The transcription factor Sox2 is a member of SRY
sub-family of HMG box transcription factors that
binds to the sequence CT/ATTG/T/AT/A and induces
DNA bending that is helpful in regulation of
transcription and chromatin architecture76. Sox2
participates in the regulation of the ICM and its
progeny or derivative cells, and is expressed in ES
KUMAR et al.: CHARACTERIZATION OF EMBRYONIC STEM CELLS
cells; but it is also expressed in neural stem cells.
Sox2 expression is associated with uncommitted
dividing stem and precursor cells of the developing
central nervous system and indeed can be used to
isolate such cells77,78. Sox2 also marks the pluripotent
lineage of the early mouse embryo, so that like Oct4,
it is expressed in the ICM, epiblast, and germ cells. Its
down-regulation correlates with a commitment to
differentiate, such that it is no longer expressed in cell
types with restricted developmental potential. Several
markers other than Oct4, nanog and sox2 have now
been realized to be exclusively expressed by ES cells.
Differentiation
Pluripotency is one of the defining features of ES
cells. Perhaps the most definitive test of pluripotency
is the formation of chimeras in mice in which ES cells
are injected into the blastocyst and the contribution of
the ES cells to the resulting chimera is assessed to
determine the differentiation capacity of the injected
cells. ES cells differentiate spontaneously in vitro in
culture grown in the absence of appropriate feeder
cells. Under the appropriate conditions, such as
suspension culture, embryiod bodies (EBs) are formed
in almost all species like canine37, sheep23, bovine42
and buffalo20,39 with region specific endoderm,
mesoderm and ectoderm differentiation26,88. The small
EBs consist of several cells1,5,11,89,90, while older EBs
look like an inverted embryo and reach 8-12 mm in
diameter.
ES cells may be directed into the lineage of interest
by supplementing various growth factors or their
antagonists into the culture media. These growth
factors or stimulating agents allow directed
differentiation of ES cells towards a particular cell
lineage or cell type (Table 3).
The differentiated cells can be identified with the
help of various markers, which are highly expressed
in these cells. Very few markers are specific for one
cell type, and as such, a panel of markers needs to be
used in order to characterize the cells (Table 4).
Epigenetic Analysis
Epigenetic mechanisms, such as histone
modifications and DNA methylation, have been
shown to play a key role in the regulation of gene
transcription. Results of recent studies indicate that a
novel bivalent chromatin structure marks key
developmental genes in ES cells wherein a number of
untranscribed lineage-control genes, such as Sox1,
Nkx2-2, Msx1, Irx3, and Pax3, are epigenetically
27
Table 3Directed differentiation of ES cells using different
growth factors and antagonists
Growth factor/antagonist
Cell type
Referen
ce
Retinoic acid
Noggin
BMP4 + bFGF
BMP2 & FGF2
Nitric oxide
TGF-B3
BMP/dexamethasoneBeta/glycerophosphate/
ascorbic acid
Insulin/triiodothyronine
TGF-B3/parathyroid
hormone
Retinoic acid + LIF +
bFGF
Neuron
Neurons/glia
Trophectoderm
91,92
91
93
94
95
96
97
Cardiomyocyte
Chondrocytes
Osteocytes
Adipocytes
Chondrocytes
Male germ cell lineage
98
Table 4Analysis of differentiated cells through markers
Germ layer
Marker
Tissue
Ectoderm
NF-68
Keratin
DbH
Brain
Skin
Adrenal
Enolase
CMP
Rennin
Kallikrein
Glut-2
cACT
bGlobin
Muscle
Bone
Kidney
Kidney
Gut
Heart
Hematopoietic
Albumin
a1-AT
PDX-1
Insulin
a-FP
Liver
Mesoderm
Endoderm
Referen
ce
68, 99)
Pancreas
Fetal liver
modified with a unique combination of activating and
repressive histone modifications that prime them for
potential activation (or repression) upon cell lineage
induction and differentiation. As is the case in the
active gene loci in differentiated somatic cell types, it
has been shown that the promoter region of active
genes in ES cells, such as Oct4 and nanog, is marked
by acetylation of H3 and H4, and that these histone
modifications are important for active gene
transcription100-102. These results indicate that ES cells
employ similar epigenetic mechanisms for active gene
transcription as compared to differentiated somatic
cells. However, recent evidences suggest that there
28
INDIAN J BIOTECHNOL, JANUARY 2009
are some unique histone modification mechanisms in
ES cells for silencing the lineage-control genes, which
are not actively transcribed in ES cells, but which
may be activated later during differentiation.
Epigenetic
modifications,
including
CpG
methylation and histone modifications regulate gene
transcription and are also important in the
maintenance of pluripotency. CpG binding protein
(CGBP) has a unique DNA-binding specificity for
unmethylated CpG dinucleotides. Mouse ES cells
lacking the CGBP show reduced levels of genomic
methylation
and
maintenance
of
DNA
methyltransferase activity. The cells remain
undifferentiated even upon LIF withdrawal103. CpG
dinucleotides of Oct3/4 and nanog genes are
hypomethylated in undifferentiated human ES cells,
whereas methylation progresses during neural
differentiation104. The methylation pattern of Oct3/4
and nanog is highly comparable with their expression
pattern, and the methylation would suppress their
transcription in differentiated cells. Thus, it has been
realized that a balance between activating and
repressive histone modifications is important for the
maintenance of pluripotency of ES cells as well as for
the cell lineage determination, and extensive studies
regarding a greater repertoire of histone modifications
in ES cells will likely to provide novel insights into
this area.
Utility of ES Cells in Farm Animals
Stem cell research provides new tools for drug
discovery and toxicology, and creates new
possibilities for understanding early development.
Besides these uses, ES cells provide a powerful tool
for the studies on early embryonic development105-107,
gene
targeting108-110,
cloning50,111,113,
chimera
114
formation
and transgenesis50. Because of their
potential use for targeted gene manipulation, isolation
of ES cells in livestock species could have enormous
agricultural and pharmaceutical applications. The use
of ES cell technology in livestock may help in
overcoming current limitations on efficient gene
transfer by providing an abundance of totipotent stem
cells to be genetically manipulated by using
conventional recombinant techniques.
Concluding Remarks
Understanding about the pluripotency of ES cells
has progressed remarkably in the last few years.
Today, there are several useful markers available for
the assessment of ES cells. However, the expression
of these markers is not same in all species. Although
ES cells appear phenotypically stable over long-term
culture in their expression of markers, ability to
differentiate, and for the maintenance of a stable
karyotype, recent studies provide evidence that
various ES cell lines may have distinct epigenetic or
developmental states. Thus, it has been suggested that
examination of the epigenetic status of ES cells
should also be included in the assessment of their
characterization. It is clear that continued
characterization of the existing and newly created ES
cells in different species will be helpful in
understanding the mechanisms involved in retaining
the undifferentiated state as well as for in depth
knowledge of mechanisms involved in differentiation.
Thus, many questions still require answer to such as
success of cloning, transgenic, using stem cell
technology. Characterization studies are of immense
potential in undifferentiated as well as differentiated
cells.
References
1
Evans M J & Kaufman M, Establishment in culture of
pluripotential cells from mouse embryos, Nature (Lond), 292
(1981) 154-156.
2
Wolf D P, Kuo H C, Pou K Y & Lester L, Progress with nonhuman primate embryonic stem cells, Biol Reprod, 71 (2004)
1766-1771.
3
Thomson J A, Itskovitzeldor J, Shapiro S S, Waknitz M A,
Swiergiel J J et al, Embryonic stem cell lines derived from
human blastocysts, Science, 282 (1998) 1145-1147.
4
Reubinoff B E, Pera M F, Fong C Y, Trounson A & Bongso
A, Embryonic stem cell lines from human blastocysts:
Somatic differentiation in vitro, Nat Biotechnol, 18 (2000)
399-404.
5
Iannaccone P M, Tabom G U, Garton R L, Caplice M D &
Brenin D R, Pluripotent embryonic stem cells from the rat
are capable of producing chimeras, Dev Biol, 163 (1994)
288-292.
6
Wheeler M B, Development and validation of swine
embryonic stem cells: A review, Reprod Fertil Dev, 6 (1994)
563-568.
7
Sukoyan M A, Votalin S Y, Golubitsa A N, Zhelezova A I,
Semenova L A et al, Embryonic stem cells derived from
morula, inner cell mass, and blastocysts of mink:
Comparisom of their pluripotencies, Mol Reprod Dev, 36
(1993) 148-158.
8
Strelchenko
N,
Bovine
pluripotent
stem
cells,
Theriogenology, 45 (1996) 131-140.
9
Saito S, Ugai H, Sawai K, Yamamoto Y, Minamihashi A et
al, Isolation of embryonic stem-like cells from equine
blastocysts and their differentiation in vitro, FEBS Lett, 531
(2002) 389-396.
10 Notarianni E, Galli C, Laurie S, Moor R M & Evans M J,
Derivation of pluripotent, embryonic cell lines from pig and
sheep, J Reprod Feriil, 43 (1991) 255-260.
KUMAR et al.: CHARACTERIZATION OF EMBRYONIC STEM CELLS
11 Graves K H & Moreadith R W, Derivation and
characterization of putative pluripotential embryonic stem
cells from preimplantation rabbit embryos, Mol Reprod Dev,
36 (1993) 424-433.
12 Thomson J A, Kalishman J, Golos T G, Durning M, Harris C
P et al, Isolation of a primate embryonic stem-cell line, Proc
Natl Acad Sci USA, 92 (1995) 7844-7848.
13 Martin G R & Evans M J, The formation of embryoid bodies
in vitro by homogeneous embryonic carcinoma cell cultures
derived from isolated single cells,. in Teratomas and
differentiation, edited by M I Sherman & D Solter
(Academic Press, New York) 1975, 169-187.
14 Bradley A, Evans M, Kaufman M H & Robertson E,
Formation of germ-line chimaeras from embryo-derived
teratocarcinoma cell lines, Nature (Lond), 309 (1984)
255-256.
15 Wobus A M, Holzhausen H, Jakel P & Schoneich J,
Characterization of pluripotent stem cell line derived from a
mouse embryo, Exp Cell Res, 152 (1984) 212-219.
16 Doetschman T C, Eistetter H, Katz M, Schmidt W & Kemler
R, The in vitro development of blastocyst derived embryonic
stem cell lines: Formation of visceral yolk sac, blood islands
and myocardium, J Embryol Exp Morphol, 87 (1985) 27-45.
17 Risau W, Sariola H, Zerwes H G, Sasse J, Ekblom P et al,
Vasculogenesis and angiogenesis in embryonic-stem cell
derived embryoid bodies, Development, 102 (1988) 471-478.
18 Hoffman L M & Carpenter M K, Characterization and
culture of human embryonic stem cells, Nat Biotechnol, 23
(2005) 699-708.
19 Familari M & Selwood L, The potential for derivation of
embryonic stem cells in vertebrates, Mol Reprod Dev, 73
(2006) 123-131.
20 Verma V, Guatam S K, Singh B, Manik R S, Palta P et al,
Isolation and characterization of embryonic stem cell-like
cells in vitro-produced buffalo (Bubalus bubalis) embryos,
Mol Reprod Dev, 74 (2007) 520-529.
21 Buzzard J J, Gough N M, Crook J M & Coleman A,
Karyotype of human ES cells during extended culturem, Nat
Biotechnol, 22 (2004) 381-382.
22 Strelchenko N, Mitalipova M & Stice S, Further
characterization of bovine pluripotent stem cells,
Theriogenology, 43 (1995) 327.
23 Dattena M, Chessa B, Lacerenza D, Accardo C, Pillichi S et
al, Isolation, culture and charecterization of embryonic cell
lines from vitrified sheep blastocysts, Mol Reprod Dev, 31
(2006) 31-39.
24 Robertson E J, Embryo-derived stem cell lines, in
Teratocarcinomas and embryonic stem cells: A practical
approach, edited by E J Robertson (IRL Press, Oxford) 1987,
71-112.
25 Resnick J L, Bixler L S, Cheng L & Donovan P J, Long-term
proliferation of mouse primordial germ cells in culture,
Nature (Lond), 359 (1992) 550-551.
26 Smith A G, Embryo-derived stem cells of mice and men,
Annu Rev Cell Dev Biol, 17 (2001) 433-462.
27 Carpenter M K, Rosler E & Rao M S, Characterization and
differentiation of human embryonic stem cells, Cloning Stem
Cells, 5 (2003) 79-88.
28 Ginis I, Luo Y, Miura T, Thies S & Brandenberger R,
Differences between human and mouse embryonic stem
cells, Dev Biol, 269 (2004) 360-380.
29
29 Talbot N C, Powell A M & Rexroad C E Jr, In vitro
pluripotency of epiblasts derived from bovine blastocysts,
Mol Reprod Dev, 42 (1995) 35-52.
30 Van Stekelenburg-Hamers A E P, Van Achterberg T A P,
Rebel H G, Felchon J E, Campbell K H S et al, Isolation and
characterization of permanent cell lines from inner cell mass
cells of bovine blastocysts, Mol Reprod Dev, 40 (1995)
444-454.
31 Stice S L, Strelchenko N, Keefer C L & Mathews L,
Pluripotent bovine embryonic stem cell lines direct
embryonic development following nuclear transfer, Biol
Reprod, 54 (1996) 100-110.
32 Cibelli J B, Stice S L, Golueke P J, Kane J J, Jerry J et al,
Transgenic bovine chimeric offspring produced from somatic
cell-derived stem-like cells, Nat Biotechnol, 16 (1998)
642-646.
33 Gjørret J O & Maddox-Hyttel P, Attempts towards derivation
and establishment of bovine embryonic stem cell-like
cultures, Reprod Fertil Dev, 17 (2005) 113-124.
34 Talbot N C, Rexroad C E, Pursel V G & Powell A M,
Alkaline phosphatase staining of pig and sheep epiblast cells
in culture, Mol Reprod Dev, 36 (1993) 139-147.
35 Li M, Zhang D, Hou Y, Jiao I, Zheng X et al, Isolation and
culture of porcine blastocyst, Mol Reprod Dev, 65 (2003)
429-434.
36 Polejaeva I A, While K, Ellis L C & Reed W A, Isolation and
long-term culture of mink and bovine embryonic stem-like
cells, Theriogenology, 43 (1995) 300.
37 Hatoya S, Torii R, Kondo Y, Okuno T, Kobayashi K et al,
Isolation and characterization of embryonic stem- like cells
from canine blastocysts, Mol Reprod Dev, 73 (2006)
298-305.
38 Kitiyanant Y, Saikhun J, Kitiyanant N, Sritanaudomchai H,
Faisaikarm T et al, Establishment of buffalo embryonic
stem-like (ES-like) cell lines from different sources of
derived blastocysts, Reprod Fertil Dev, 16 (2004) 216.
39 Chauhan M S, Verma V, Manik R S, Palta P, Singla S K et
al, Development of inner cell mass and formation of
embryoid bodies on a gelatin-coated dish and on the feeder
layer in buffalo (Bubalus bubalis), Reprod Fertil Dev, 18
(2006) 205-206.
40 Hamano S, Watanabe Y, Azumi S & Toyoda Y,
Establishment of embryonic stem (ES) cell-like cell lines
derived from bovine blastocysts obtained by in vitro culture
of oocytes matured and fertilized in vitro, J Reprod. Dev, 44
(1998) 297-303.
41 Wang L, Duan E, Sung L Y, Jeong B S, Yang X et al,
Generation and characterization of pluripotent stem cells
from cloned bovine embryos, Biol Reprod, 73 (2005)
149-155.
42 Yadav P S, Wilfried A K, Herrmann D, Carnwath J W &
Niemann H, Bovine ICM derived cells express Oct4
ortholog, Mol Reprod Dev, 72 (2005) 182-190.
43 Gooi H C, Feizi T, Kapadia A, Knowles B B, Solter D et al,
Stage-specific embryonic antigen involves α1-3 fucosylated
type 2 blood group chains, Nature (Lond), 292 (1981) 156158.
44 Solter D & Knowles B B, Monoclonal antibody defining a
stage-specific mouse embryonic antigen (SSEA-1), Proc
Natl Acad Sci USA, 75 (1978) 5565-5569.
30
INDIAN J BIOTECHNOL, JANUARY 2009
45 Matsui Y, Zsebo K & Hogan B L M, 1992. Derivation of
pluripotential embryonic stem cells from murine primordial
germ cells in culture, Cell, 70 (1992) 841-847.
46 Gompert M, Garcia-Castro M, Wylie C & Heasman J,
Interactions between primordial germ cells plays a role in
their migration in mouse embryos, Development, 120 (1994)
135-141.
47 Henderson J K, Draper J S, Baillie H S, Fishel S, Thomson J
A et al, Preimplantation human embryos and embryonic stem
cells show comparable expression of stage-specific
embryonic antigens, Stem Cells, 20 (2002) 329-337.
48 Thomson J A & Marshal V S, Primate embryonic stem cells,
Curr Top Dev Biol, 38 (1998) 133-165.
49 Mitalipova M, Beyhan Z & First N L, Pluripotency of bovine
embryonic cell line derived from precompacting embryos,
Cloning, 3 (2001) 59-67.
50 Saito S, Sawaki K, Ugai H, Moriyasu S & Minamihashi A,
Generation of cloned calves and transgenic chimeric
embryos from bovine embryonic stem-like cells, Biochem
Biophys Res Commun, 309 (2003) 104-113.
51 Shevinsky L H, Knowles B B, Damjanov I & Solter D,
Monoclonal antibody to murine embryos defines a stagespecific embryonic antigen expressed on mouse embryo and
human teratocarcinoma cells, Cell, 30 (1982) 697-705.
52 Kannagi R, Cochran N A, Ishigami F, Hakamori S I,
Andrews P W et al, Stage-specific embryonic antigens
(SSEA-3 and -4) are epitopes of a unique globoseries
ganglioside isolated from human teratocarcinoma cells,
EMBO J, 2 (1983) 2355-2361.
53 Andrews P W, Damjanov I, Simon D, Banting G S, Carlin C
et al, Pluripotent embryonal carcinoma clones derived from
the human teratocarcinoma cell line Tera-2, Lab Invest, 50
(1984) 147-162.
54 Liu S, Liu H, Tang S, Pan Y, Ji K et al, Characterization of
stage-specific embryonic antigen-1 expression during early
stages of human embryogenesis, Oncol Rep, 12 (2004)
1251-1256.
55 Andrews P W & Knowles B B, Human teratocarcinoma in
Teratocarcinoma and embryonic cell interactions, edited by
T Muramatsu, G Gachelin, A A Moscona & Y Ikawa (Japan
Scientific Societies Press and Academic Press, Tokyo) 1982,
19-30.
56 Draper J S, Pigott C, Thomson J A & Andrews P W, Surface
antigens of human embryonic stem cells: changes upon
differentiation in culture, J Anatomy, 200 (2002) 249-258.
57 Sritanaudomachi H, Pavasuthipasit K, Kitiyanant Y,
Kupradinum P, Mitalipova S et al, Characterization and
multilenease differentiation of embryonic stem cells derived
from a buffalo parthenogenetic embryo, Mol Reprod Dev, 74
(2007) 1295-1302.
58 Badcock G, Pigott C, Geopel J & Andrews P W, The human
embryonal carcinoma marker antigen TRA-1-60 is a
sialylated keratin sulfate proteoglycan, Cancer Res, 59
(1999) 4715-4719.
59 Amit M, Shariki C, Margulets V & Itskovitz-Eldor J, Feeder
layer and serum free culture of human embryonic stem cells,
Biol Reprod, 70 (2004) 837-845.
60 Pera M F, Reubinoff B & Trounson A, Human embryonic
stem cells, J Cell Sci, 113 (2000) 5-10.
61 Mikkola M, Olsson C, Palgi J, Ustinov J, Palomaki T et al,
Distinct differentiation characteristics of individual human
embryonic stem cell lines, Dev Biol, 6 (2006) 40.
62 Nichols J, Zevnik B, Anastassiadis K, Niwa H, KleweNebenius D et al, Formation of pluripotent stem cells in the
mammalian embryo depends on the POU transcription factor
Oct4, Cell, 95 (1998) 379-391.
63 Scholer H R, Dressler G R, Balling R, Rohdewohld H &
Gruss P, Oct-4: A germline-specific transcription factor
mapping to the mouse t-complex, EMBO J, 9 (1990)
2185-2195.
64 Avilion A A, Nicolis S K, Pevny L H, Perez L, Vivian N
et al, Multipotent cell lineages in early mouse development
depend on SOX2 function, Genes Dev. 17 (2003) 126-140.
65 Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M
et al, The homeoprotein Nanog is required for maintenance
of pluripotency in mouse epiblast and ES cells, Cell, 113
(2003) 631-642.
66 Chambers I, Colby D, Robertson M, Nichols J, Lee S et al,
Functional expression cloning of Nanog, a pluripotency
sustaining factor in embryonic stem cells, Cell, 113 (2003)
643-655.
67 Hart A H, Hartley L, Ibrahim M, & Robb L, Identification,
cloning and expression analysis of the pluripotency
promoting Nanog genes in mouse and human, Dev Dyn, 230
(2004) 187-198.
68 Lee J B, Song J M, Lee J E, Park J H, Kim S J et al,
Available human feeder cells for the maintenance of
embryonic stem cells, Reproduction, 128 (2004) 727-735.
69 Scholer H R, Balling R, Hatzopoulos A K, Suzuki N & Gruss
P, A family of octamer- specific protein present during
mouse embryonesis: Evidence for germ line-specific
expression of an Oct factor, EMBO J, 8 (1989) 2543-2550.
70 Rosner M H, Vigano M A, Ozato K, Timmons P M, Poirier F
et al, A POU-domain transcription factor in early stem cells
and germ cells of the mammalian embryo, Nature (Lond),
345 (1990) 686-692.
71 Okamoto K, Okazawa H, Okula A, Sakai M, Muramatsu M
et al, A novel octamer binding transcrition factor is
differentially expressed in mouse embryonic stem cells, Cell,
60 (1990) 461-472.
72 Donovan P & Miguel M P, Turnig germ cell into stem cells,
Curr Opin Genet Dev, 13 (2003) 463-471.
73 Van Eijk M J, Van Rooijen M A, Modina S, Scesi L, Folkers
G et al, Molecular cloning, genetic mapping and
developmental expression of bovine POU5F1, Biol Reprod,
60 (1999) 1093-1103.
74 He S, Pant D, Bischoff S, Gavin W, Melican D et al,
Expression of pluripotency - deretmining factors Oct-4 and
Nanog in preimplantiation goat embryos, Reprod Fertil Dev,
17 (2004) 203.
75 Wang X, Akkari Y, Willenbring H, Torimaru Y, Foster M
et al, Cell fusion is the principal source of bone- marrowderived hepatocytes, Nature (Lond), 422 (2003) 897-901.
76 Pevny L H & Lovell-Badge R, Sox genes find their feet,
Curr Opin Genet Dev, 7 (1997) 338-344.
77 Li M, Pevny L, Lovell-Badge R & Smith A, Generation of
purified neural precursors from embryonic stem cells by
lineage selection, Curr Biol, 8 (1998) 971-974.
78 Zappone M V, Galli R, Catena R, Meani N, De Biasi S et al,
Sox2 regulatory sequences direct expression of a-geo
KUMAR et al.: CHARACTERIZATION OF EMBRYONIC STEM CELLS
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
transgene to telencephalic neural stem cells and precursors of
the mouse embryo, revealing regionalization of gene
expression in CNS stem cells, Development, 127 (2000)
2367-2382.
Kirchhof N, Carnwath J W, Lemme E, Anastassiadis K,
Scholer H et al, Expression pattern of Oct-4 in
preimplantation embryos of different species, Biol Reprod,
69 (2000) 1698-1705.
Kurosaka S, Eckhardt S & Mclaughlin K J, Pluripotent
lineage defetition in bovine embryos by oct4 transcript
localization, Biol Reprod, 71 (2004) 1578-1582.
Rodda D, Chew J L, Lim L H, Loh Y H, Wang B et al,
Transcriptional regulation of Nanog by Oct4 and Sox2,
J Biol Chem, 280 (2005) 24731-24737.
Carlin R, Davis D, Weiss M, Schultz B & Troyer D,
Expression of early transcription factoers Oct-4, Sox-2 and
Nanog by procine umbilical cord (PUC) matrix cells,
Reprod. Biol Endocrinol, 4 (2006) 8.
Sperger J M, Chen X, Draper J S, Antosiewicz J E, Chon C
H et al, Gene expression patterns in human embryonic stem
cells and human pluripotent germ cell tumours, Proc Natl
Acad Sci USA, 100 (2003) 13350-13355.
Abeyta M J, Clark A T, Rodriguez R T, Bodnar M S,
Pera R A et al, Unique gene expression signatures of
independently-derived human embryonic stem cell lines,
Hum Mol Genet, 13 (2004) 601-608.
Ramalho-Santos M, Yoon S, Matsuzaki Y, Mulligan R C &
Melton D A, ‘‘Stemness’’: Transcriptional profiling of
embryonic and adult stem cells, Science, 298 (2002)
597-600.
Tanaka T S, Kunath T, Kimber W L, Jaradat S A, Stagg C A
et al, Gene expression profiling of embryo-derived stem cells
reveals candidate genes associated with pluripotency and
lineage specificity, Genome Res, 12 (2002) 1921-1928.
D’Amour K A & Gage F H, Genetic and functional
differences between multipotent neural and pluripotent
embryonic stem cells, Proc Natl Acad Sci USA, 100 (2003)
11866-11872.
Keller G, In vitro differentiation of embryonic stem cells,
Curr Opin Cell Biol, 7 (1995) 862-869.
Eistetter H R, Pluripotent embryonal stem cell lines can be
established from disaggregated mouse morulae, Dev Growth
Diff, 31 (1989) 275-282.
Giles J R, Yang X, Mark W & Foote R I-I, Pluripotency of
cultured rabbit inner cell mass cells detected by isozyme
analysis and eye pigmentation of fetuses following injection
into blastocysts or morulae, Mol Reprod Dev, 36 (1993)
130-138.
Pera M F, Andrade J, Houssami S, Reubinoff B, Trounson A
et al, Regulation of human embryonic stem cell
differentiation by BMP-2 and its antagonist noggin, J Cell
Sci, 117 (2004) 1269-1280.
Verani R, Cappucio I, Spinsanti P, Grandini R, Caruso A
et al, Expression of the Wnt inhibitor Dickkop f-1 is required
for the induction of neural markers in mouse embryonic stem
cells differentiating in response to retinoic acid, J
Neurochem, 100 (2007) 242-250.
Xu R H, Chen X, Li R, Addicks G C, Glennon C et al,
BMP4 initiate human embryonic stem cell differentiation to
trophoblast, Nat Biotechol, 20 (2002) 1261-1264.
31
94 Kawai T, Takahashi T, Esaki M, Ushikoshi H, Nagona S
et al, Efficient cardiomyogenic differntiation of embryonic
stem cell by fibroblast growth factor 2 and bone
morphogenetic protein 2, Circ J, 68 (2004) 691-702.
95 Kanno S, Kim P K, Sallam K, Lei J, Billiar T R et al, Nitric
oxide facilitates cardiomyogenesis in mouse embryonic stem
cells, Proc Natl Acad Sci, 98 (2004) 10716-10721.
96 Kawaguchi J, Mee P J & Smith A G, Osteogenic and
chondrogenic differentiation of embryonic stem cells in
response to specific growth factors, Bone, 36 (2005)
758-769.
97 Kawaguchi J, Generation of osteoblasts and chondrocytes
from embryonic stem cells, Methods Mol Biol, 330 (2006)
135-148.
98 Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K et al,
Derivation of embryonic germ cells and male gametes from
embryonic stem cells, Nature (Lond), 427 (2004) 148-154.
99 Yoo S J, Yoon B S, Kim J M, Song J M, You S et al,
Efficient culture system for human embryonic stem cells
using autologous human embryonic stem cell- derived feeder
cells, Exp Mol Med, 37 (2005) 399-407.
100 Hattori N, Nishino K & Ko Y G, Epigenetic control of
mouse Oct-4 gene expression in embryonic stem cells and
trophoblast stem cells, J Biol Chem, 279 (2004)
17063-17069.
101 Kimura H, Tada M & Nakatsuji N, Histone code
modifications on pluripotential nuclei of reprogrammed
somatic cells, Mol Cell Biol, 24 (2004) 5710-5720.
102 O’Neill L P, VerMilyea M D & Turner B M, Epigenetic
characterization of the early embryo with a chromatin
immunoprecipitation protocol applicable to small cell
populations, Nat Genet, 38 (2006) 835-841.
103 Carlone D L, Lee J H & Young S R, Reduced genomic
cytosine methylation and defective cellular differentiation in
embryonic stem cells lacking CpG binding protein, Mol Cell
Biol, 25 (2005) 4881-4891.
104 Deb-Rinker P, Ly D, Jezierski A, Sikorska M & Walker P R,
Sequential DNA methylation of the Nanog and Oct-4
upstream regions in human NT2 cells during neuronal
differentiation, J Biol Chem, 280 (2005) 6257-6260.
105 Robertson E J, Using embryonic stem cells to introduce
mutations into the mouse germ line, Biol Reprod, 44 (1991)
238-245.
106 Wiles M V & Johansson B M, Analysis of factors controlling
primary germ layer formation and early hematopoiesis using
embryonic stem cell in vitro differentiation, Leukemia, 11
(1997) 454-456.
107 Rathjen J, Lakes J A, Bettes M D, Washington J M,
Chapman G et al, Formation of primitive ectoderm-like cell
population, EPL cells; from ES cell in response to biological
derived factors, J Cell Sci, 112 (1999) 601-612.
108 Lohnes D, Gene targeting of retinoid receptors, Mol
Biotechnol, 11 (1999) 67-84.
109 Jakobs P M, Smith L, Thayer M & Grompe M, Embryonic
stem cells can be used to conduct hybrid cell lines containing
a single, selectable murine chromosome, Mammalian
Genome, 10 (1999) 381-384.
110 Zheng B, Mills A A & Bradley A, A system for rapid
regeneration of coat colour-tagged knockouts and defined
32
INDIAN J BIOTECHNOL, JANUARY 2009
chromosomal rearrangements in mice, Nucleic Acids Res, 27
(1999) 2354-2360.
111 Sims M & First N L, Production of calves by transfer of
nuclei from cultured inner cell mass cells, Proc Natl Acad
Sci USA, 90 (1993) 6143-6147.
112 Keefer C L, Stice S L & Matthews D L, Bovine inner cell
mass cells as donor nuclei in the production of nuclear
transfer embryos and calves, Biol Reprod, 50 (1994)
935-939.
113 Iwasaki S, Campbell K H, Galli C & Akiyama K, Production
of live calves derived from embryonic stem cell like cells
aggregated with tetraploid embryos, Biol Reprod, 62 (2000)
470-475.
114 Butler J E, Anderson G B, BonDurant R H, Pashen R L &
Penedo M C T, Production of ovine chimeras by inner cell
mass transplantation, J Anim Sci, 65 (1987) 317-324.