Insights into Embryonic Stem Cells of Bovines
Embryonic Stem (ES) cells are derived from blastocyst and these cells have the capability to generate all embryonic tissues in vitro. This propensity of ES cells has acquired considerable attention in recent years due to the promising potential for future cell replacement-based therapies. The in vitro differentiation capacity of ES cells provides unique opportunities for experimental analysis of gene regulation and function during cell commitment and differentiation in early embryogenesis. The ES cells are pluripotent cell lines with the capacity of self-renewal and a broad differentiation plasticity. They are derived from pre-implantation embryos and can be propagated as a homogeneous, uncommitted cell population for an almost unlimited period of time without losing their pluripotency and their stable karyotype. The ES cell technology is of high interest for researchers associated with livestock species. Simultaneously, research activities are being focused on characteristics and differentiation potential of Somatic Stem Cells (SSCs), unraveling an unexpected plasticity of these cell types. Somatic stem cells are found in differentiated tissues and can renew themselves in addition to generating the specialized cell types of the tissue from which they originate. Additional to discoveries of SSCs in tissues that were previously not thought to contain these kinds of cells, they also appear to be capable of developing into cell types of other tissues, but have a reduced differentiation potential as compared to embryo-derived stem cells. Therefore, SSCs are referred to as multipotent rather than pluripotent. This review summarizes characteristics of pluripotent ESCs in bovines and evaluates their potentials for in vitro propagation and differentiation as well as their potential uses in cell based therapies.
March 25, 2010; Accepted: May 02, 2010;
Published: July 27, 2010
Embryonic Stem Cells (ESCs) are unique pluripotent cells derived from pre-implantation
blastocyst-stage embryos. They can undergo asymmetric division whereby they
either duplicate themselves or differentiate into another cell-type. While adult
stem cells are multipotent and can only differentiate into a limited number
of cell-types, ESCs are capable of differentiating into any cell-type. ESCs
can proliferate indefinitely in an undifferentiated state (Evans
and Kaufman, 1981). They express specific markers or characteristics including
stage specific embryonic antigens, enzymatic activities such as alkaline phosphatase
and telomerase and stemness genes that are rapidly down-regulated upon differentiation,
including Oct4 and Nanog (Byrne et al., 2006).
Alternatively, they can differentiate in vivo in teratomas into cells
representing the three major germ layers: endoderm, mesoderm or ectoderm and
they can be directed to differentiate in vitro into any of the more than
200 cell types present in the adult body (Savatier et
al., 1994). Another characteristic of ESCs is their apparent ability
to maintain a normal karyotype through large passage numbers. Indeed, a feature
of primary cell cultures is the development of abnormal karyotypes as they become
senescent. Since many human diseases result from defects in a single cell type,
the potential to replace defective cells by cell or tissue replacement therapy
involving differentiated human ESCs (hESCs) provides a possible cure for, or
at least the alleviation of symptoms of, various degenerative diseases (Srivastava
and Sejian, 2010).
The ESCs promise to open a new window in human existence. They possess the
unique potential to replace our cells as they age, mutate and die. This scientific
advance offers us the tantalizing possibility of maintaining our bodies in a
state of mental and physical well-being inconceivable even one generation ago.
As we enter the new millennium, the gift we are being offered is nothing short
of the chance at longer, healthier lives. Stem cell based therapies for the
repair and regeneration of various tissues and organs offer a paradigm shift
that may provide alternative therapeutic solution for many diseases. Although,
ESCs and Induced Pluripotent Stem Cells (iPSC) are theoretically highly beneficial,
there are various limitations to their use imposed by cell regulation, ethical
consideration and genetic manipulation (Cauffman et al.,
2005). Adult Stem Cells (ASCs) on the other hand, are more easily available
with neither ethical nor immunoreactive considerations, as long as they are
of autologous tissue origin. In recent years much research has been focused
on Mesenchymal Stem Cells (MSc) isolation from bone marrow stroma which have
been shown to possess adipogenic, osteogenic, chondrogenic, myogenic and neurogenic
potentials in vitro (Woodbury et al., 2000).
As ESCs offers wide opportunity for therapeutic potential in animals, this review
collates and synthesizes literature on characteristics of pluripotent ESCs in
bovines and evaluates their potentials for in vitro propagation and differentiation
as well as their potential uses in cell based therapies.
EPIGENETICS OF ESCs
Mammalian development originates from a single cell (zygote) that upon cleavage
gives rise to totipotent blastomeres of the early embryo that eventually proliferate
and differentiate into the wide variety of cell phenotypes found in the adult
body. The complex pattern of gene expression governing development and differentiation
is tightly regulated by epigenetic modifications, i.e., modifications of chromatin
not involving changes in the DNA sequence. DNA methylation and histone methylation/acetylation
are well known examples of epigenetic modifications. In general, DNA methylation
is associated with the silencing of gene expression. Epigenetic errors can arise
randomly or under the influence of the environment and often result in disease
in humans. For example, DNA methylation has become increasingly implicated in
cancer, as many cancer cells contain hypermethylated DNA that in turn can lead
to the silencing of tumor suppressor genes by promoter methylation (Weksberg
et al., 2003). Genomic imprinting is a form of the epigenetic program
that involves modification of a gene or a chromosomal region that results in
absolute or preferential, monoallelic-expression of a specific parental allele.
Imprinting genes tend to cluster in the genome. Figure 1 describes
the derivation potentials of embryonic stem cells.
|| Derivation potentials of embryonic stem cells
KEY CHARACTERISTICS OF ESCs
The ESCs are distinguished by two distinctive properties: their pluripotency
and their capability to self-renew themselves indefinitely (Ying
et al., 2003). ESCs are pluripotent, that is, they are able to differentiate
into all derivatives of the three primary germ layers: ectoderm, endoderm and
mesoderm. These include each of the more than 220 cell types in the adult body.
Pluripotency distinguishes embryonic stem cells from adult stem cells found
in adults; while embryonic stem cells can generate all cell types in the body,
adult stem cells are multipotent and can only produce a limited number of cell
types. Additionally, under defined conditions, ESCs are capable of propagating
themselves indefinitely. This allows ESCs to be employed as useful tools for
both research and regenerative medicine, because they can produce limitless
numbers of themselves for continued research or clinical use. Because of their
plasticity and potentially unlimited capacity for self-renewal, ESC therapies
have been proposed for regenerative medicine and tissue replacement after injury
or disease. Diseases that could potentially be treated by pluripotent stem cells
include a number of blood and immune-system related genetic diseases, cancers
and disorders; juvenile diabetes; parkinson's; blindness and spinal cord injuries.
Besides the ethical concerns of stem cell therapy, there is a technical problem
of graft-versus-host disease associated with allogeneic stem cell transplantation.
However, these problems associated with histocompatibility may be solved using
autologous donor adult stem cells, therapeutic cloning, stem cell banks or more
recently by reprogramming of somatic cells with defined factors (e.g., induced
pluripotent stem cells).
|| Embryonic stem cell characteristics
Other potential uses of ESCs include investigation of early human development,
study of genetic disease and as in vitro systems for toxicology testing.
Figure 2 describes the key characteristics of ESCs.
STEM CELL LINEAGES
To ensure self-renewal, stem cells undergo two types of cell division. Symmetric
division gives rise to two identical daughter cells both endowed with stem cell
properties. Asymmetric division, on the other hand, produces only one stem cell
and a progenitor cell with limited self-renewal potential. Progenitors can go
through several rounds of cell division before terminally differentiating into
a mature cell. It is possible that the molecular distinction between symmetric
and asymmetric divisions lies in differential segregation of cell membrane proteins
(such as receptors) between the daughter cells. An alternative theory is that
stem cells remain undifferentiated due to environmental cues in their particular
niche. Stem cells differentiate when they leave that niche or no longer receive
those signals. Studies in 'Drosophila germarium have identified the signals
dpp and adherens junctions that prevent germarium stem cells from differentiating.
The signals that lead to reprogramming of cells to an embryonic-like state are
also being investigated. These signal pathways include several transcription
factors including the oncogene c-Myc (Vackova et al.,
2007). Initial studies indicate that transformation of mice cells with a
combination of these anti-differentiation signals can reverse differentiation
and may allow adult cells to become pluripotent. However, the need to transform
these cells with an oncogene may prevent the use of this approach in therapy.
The ESC lines have been successfully isolated from mouse, monkey and human
blastocysts, although outstanding derivations have also been made using embryos
at pre-compaction stages (Eistetter, 1988; Delhaise
et al., 1996; Strelchenko, 1996; Mitalipova
et al., 2001). Most attempts to isolate and culture bESCs have been
done with day 7-9 bovine blastocysts (Stice et al.,
1996; Strelchenko, 1996; Cibelli
et al., 1998; Iwasaki et al., 2000;
Betts et al., 2001; Saito
et al., 2003; Roach et al., 2006;
Munoz et al., 2008) although, ESC-like cells
are also isolated from day 12-14 embryos (Gjorret and Maddox-Hyttel,
2005). Yet, the optimal timing of bovine pre-implantation development to
derive ESCs is still unknown. Attempts to derive bESC from zygotes and early
cleavage stage embryos mostly failed (Strelchenko, 1996;
Mitalipova et al., 2001), while only a single
bovine embryonic cell line, generated from a two-cell embryo, has been cultured
over 3 years (Mitalipova et al., 2001). Yet,
when bovine morulae were used as starting material, efficient colony formation
rates ranged over 60-70% (Stice et al., 1996;
Strelchenko, 1996). The embryonic stage (morulae and
day 7 blastocysts) used did not influence the efficiency in establishing bESC
colonies. These results are nevertheless contrary to studies reporting that
Day-8 hatched blastocysts yield a higher proportion of epiblast colonies than
Inner Cell Masses (ICMs) isolated from day 9 blastocysts (41 and 13%, respectively)
(Talbot et al., 1995).
Bovine embryos from different sources have been used to isolate bESCs (Meenambigai
et al., 2010; Stalin et al., 2010).
Yet there is only one published report which aimed to compare the feasibility
of in vitro and in vivo derived embryos for the isolation of
pluripotent cells (Talbot et al.,1995). This
study demonstrated that in vivo derived (IVD) blastocysts, especially
from early hatching blastocysts, were shown to be a source of pluripotent epiblasts
superior to their in vitro produced (IVP) counterparts. The basis for
any advantage by in vivo produced blastocyst to produce ESC lines is
not known, although, a number of differences in morphology, metabolic rates,
gene expression and susceptibility to cooling damage (Smith
et al., 2005; Lonergan et al., 2006)
have been reported between IVD and IVP bovine embryos. It is possible that the
reduced number of cells present in the ICM of IVP bovine embryos (Van
Soom et al., 1996) might affect survival of the ICM in culture, hindering
the chances to establish ESC lines from IVP embryos. In fact Anderson
et al. (1994) assumed that the factor that may affect survival of
porcine ICMs in culture was the number of cells of the ICMs. In vivo
derived embryos might be a better source of pluripotent cells, but their use
as a starting material to isolate ESC is expensive and laborious (Meenambigai
et al., 2009; Prabha et al., 2010).
Therefore, it would be advisable to improve the procedures to derive ESCs from
IVP embryos, as well as the ability of IVPICMs to yield ESCs. An obvious way
to progress in this aspect would be increasing numbers of cells in these ICMs.
DERIVATION AND CULTURE CONDITIONS FOR ESC
Table 1 depicts the different types of cell culture media
for culturing embryonic stem cells. Culture conditions close to those established
for murine ESC culture were successfully used to derive monkey (Thomson
et al., 1995) and human ESCs (Thomson et al.,
1998). Nevertheless, it soon became evident that some factors required for
the maintenance of mESC pluripotency were not only dispensable in maintaining
hESC pluripotency but were also detrimental. As an example, this occurred with
BMP4, a member of the transforming growth factor-b (TGF-b) family involved in
controlling mESC differentiation that induces differentiation of human ESCs
into trophoblast cells (Xu et al., 2002). Since
then, considerable amount of data have been published over differences between
mouse and human pluripotency maintaining factors and signaling pathways (Renard
et al., 2007) (Fig. 3). Until now, following a
similar approach to primate ESC isolation, most attempts to culture bESC have
been inspired by the original culture methods for mESC of Evans
and Kaufman (1981).
|| Different types of cell culture media for culturing embryonic
|bFGF: Basic fibroblast growth factor, b-ME: b-mercaptoethanol,
DMEM: Dulbeccos Modified Eagles Medium, FBS: Foetal bovine serum;
FCS: Foetal calf serum, hEGF: Human epidermal growth factor, hLIF: Human
leukaemia inhibitory factor; MEM: Minimum essential medium
|| Multipotential differentiation characteristics of embryonic
Bovine ESCs are usually cultured on mouse embryonic fibroblasts (primary MEF
or transformed STO cells). Culture media consists of Dulbeccos Modified
Eagles Medium supplemented with foetal bovine serum, L-glutamine, 2-b
mercaptoethanol and different growth factors, mostly Leukaemia Inhibitory Factor
(LIF) and Epidermal Growth Factor (EGF) (for comparison of some bESC culture
conditions (Table 1). Yet it is likely that culture conditions
suitable to maintain mESC could be inadequate to maintain undifferentiated bESC.
Preliminary studies by Keefer et al. (2007) showed
that the bovine ICM and its primary outgrowths express the LIF receptor and
gp130 transducer. Yet, LIF did not improve the establishment and maintenance
of ESCs from other ungulates (Vackova et al., 2007)
although its presence in pig ESC culture medium prevented EB formation (Brevini
et al., 2007). It can be speculated that, such as in hESC, stimulation
of the STAT3 pathway by LIF might not induce proliferation of ungulate ESCs.
Similarly, some growth factors such as TGB-b, EGF or insulin-like growth factors (IGFs) found to suppress differentiation of mESCs ] did not inhibit differentiation of porcine is one of the possible explanations for the failure to isolate these cells. Identification of specific pluripotency signalling pathways will help to determine which growth factors are beneficial or which ones are inappropriate for establishing a successful bESC culture. Figure 3 describes the multipotential differentiation characteristics of ESCs.
SIMPLIFIED SCHEME OF STEM CELL POPULATIONS
Particular stem cell types are classified based on their differentiation potentials.
The zypote and morula stages can give rise to both embryonic and extra embryonic
tissues and hence can generate a complete embryo. The three germ layers, as
well as embryonic germ cells, originate from the ESCs from the inner cell mass
of the blastocysts. Adult stem cells produce progenitor cells and differentiated
tissue. Figure 4 describes the differentiation potentials
of stem cell populations.
EXPRESSION PROFILING OF ESC
Morphology, as well as the capacity to differentiate in vitro through
Embryoid Body (EB) formation, was one of the two defining criteria initially
used to identify bESC cultures. Other traits such as small size, rounded shape
or high nucleus to cytoplasm ratio were used to define bESC lines. Yet, cells
belonging to trophectoderm (TE) and visceral endoderm, which usually can be
found in blastocysts or isolated ICMs primary cultures, may be confounded with
ESC if solely morphological features are used as evaluating criteria. Bovine
blastocyst-derived TE and endoderm cell lines have been thoroughly characterized
not only by morphological criteria but also by the expression tissue-specific
marker. For instance, transferrin is a definitive marker for bovine blastocyst-derived
endoderm cell lines (Talbot et al., 2000). Therefore,
the combined use of morphological criteria and the analysis of extra-embryonic
markers is suggested to truly identify bESC and/or rule out the presence of
TE or visceral endoderm cells in ESC cultures. A useful strategy to characterize
ESC lines is to analyze the expression of pluripotency-related molecular markers.
Unfortunately, until now, no specific markers have been identified in bovine.
Therefore, markers associated to pluripotency in other species (heterospecific
pluripotency markers) such as stage-specific embryonic antigens (SSEA-1, -3,
-4) have been used to characterize bESC. SSEAs are developmentally regulated
cell surface antigens expressed by murine and human pluripotent cells (Fig.
||Differentiation potentials of stem cell populations. Particular
stem cell types are classified based on their differentiation potentials.
The zygote and morula stages can give rise to both embryonic and extra embryonic
tissues and hence can generate a complete embryo. The three germ layers,
as well as embryonic germ cells, originate from the embryonic stem cells
from the inner cell mass of the blastocysts. Adult stem cells produce progenitor
cells and differentiated tissue. Figure modified after keller
The mESCs strongly express SSEA-1 (Solter and Knowles,
1978; Gooi et al., 1981), whereas, differentiated
mESCs are characterized by the loss of SSEA-1 expression and in some instances,
by the appearance of SSEA-3 and SSEA-4 (Solter and Knowles,
In contrast, hESCs typically express SSEA-3 and SSEA-4, but not SSEA-1 and
their differentiation is characterized by down-regulation of SSEA-3 and SSEA-4
and up-regulation of SSEA-1 (Andrews et al., 1984;
Fenderson et al., 1987). Undifferentiated hESCs
also express the keratin sulphate-associated antigens TRA-1-60 and TRA-1-81
(Andrews et al., 1984). In bovine, a positive
staining for SSEA-1, SSEA-3 and SSEA-4 was reported in three embryonic cell
lines derived from pre-compaction embryos (Mitalipova et
al., 2001). Similarly, SSEA-1 expression was also detected by Saito
et al. (2003), while none of the bovine ES-like cells analysed by
these authors were found positive for SSEA-3 or SSEA-4. In contrast, Wang
et al. (2005) reported a positive SSEA-4 staining in the absence
of SSEA-1 staining in five ESC lines. Munoz et al.
(2008) reported positive staining for SSEA-4, TRA- 1-60 and TRA-1-81 in
Unfortunately, the above antigens were not only present in the ICM of bovine
blastocysts but also in the TE (Fig. 1). Therefore, in bovine,
these markers are not specific for undifferentiated and/or pluripotent cells.
The use of such markers to characterize bESC may mislead researchers into isolating
and culturing TE derived cells instead of ESCs. The expression of SSEAs, in
the TE of bovine blastocysts, was unexpected, considering that for a long time
SSEAs have been used to characterize undifferentiated bESCs. Nevertheless, it
was not totally surprising as bovine TE cells show a slow differentiating phenotype
characterized by the co-expression of epiblast-specifying genes (OCT-4, SOX-2,
NANOG) and proteins (OCT-4, NANOG) and trophoblast-specific genes (CDX-2, HAND1,
ETS-2, IFN-TAU, C12) (Kirchhof et al., 2000;
Degrell et al., 2005; Munoz et al., 2008).
Therefore in bovine, the expression of markers which are associated to pluripotency
in other species (SSEA-4, TRA-1-60, TRA-1-81, OCT-4, NANOG) is not restricted
to pluripotent cells (Fig. 1, 2), which
is a warning to validate any pluripotency marker before its heterospecific use.
An additional difficulty to characterize bESC is that available antibodies currently
used to characterize ESCs are produced using mouse or human proteins as immunogens.
Therefore, their ability to cross-react with the appropriated bovine protein
should be evaluated before their use.
It is generally, assumed that ESC biology is regulated through transcriptional
mechanisms, but the definition of a stem cell remains largely functional. The
developmental capacity of ESC lines requires a set of genes that are not expressed
in other cell types and knowledge of the intricate mechanisms regulating ESC
pluripotentiality and differentiation potential is currently limited to a few
signaling pathways (e.g., LIF, BMP, Wnt) and regulatory factors (e.g., Oct-3/4,
Nanog) (Munoz et al., 2008). Theoretically, a
comprehensive analysis of a cellular transcriptome (i.e., all the RNAs present
in a cell type) should be sufficient to define the molecular phenotype of stem
cells and establish the determinants of ESC choice. The underlying hypothesis
behind these assumptions suggests that some mRNAs will be uniquely or more abundantly
expressed in embryonic and/or adult stem cells than in any other cell type and
that comparisons among cell populations will reveal these differences. Although
several transcriptome-based (microarrays or SAGE) studies have now been published,
which claim to have identified potential stemness-associated factors, a closer
inspection of the data indicates that the identification of stemness factors
has proved elusive (Byrne et al., 2006). This
is true for both mouse and human ESCs. The reasons most frequently cited for
variations among studies include cell lines, culturing conditions, array and
hybridization protocols, data analysis and potentially contaminating cells.
Additionally, many of the studies in mice focused on comparisons among ESCs
with adult stem cells, because of earlier studies suggesting a broader potential
or plasticity of ASCs than previously believed (Niimi et
al., 2005); however, this broader plasticity of primary isolates of
many adult stem cells has recently been called into question (Niimi
et al., 2005). The identification of stemness genes by these approaches,
therefore, remains the topic of lively debate and much conjecture. Finally,
the phenotype of ESCs must also involve complex processes that alter protein
abundance both as a consequence of gene activation and processing (transcription,
splicing, etc.), as well as regulatory events associated with translation and
Post Translational Modifications (PTM). Proteomic approaches are therefore required
to visualize and interpret the phenotype of undifferentiated ESCs. Figure
5 describes the combinatorial signaling pathways in maintaining mouse ESC
MOLECULAR STRATEGY FOR ESC IDENTIFICATION
The pluripotent properties of ESCs are the basis of gene targeting technologies
used to create mutant mouse strains (transgenic and knockout) for gene function
studies in vivo. The in vitro differentiation of ESCs allows investigators
to study cell differentiation and gene regulation, as well as pharmacological
effects on functionally active cells (e.g., cardiomyocytes). Potentially, the
greatest importance is to establish strategies for cell therapy and gene therapy.
The differentiation of ESC can also provide a very useful system for the identification
of genes involved in the development of a specific cell line. Commonly used
methods including hanging drop and culture in suspension, result in the initial
formation of multi-ESC aggregates and further differentiation into EBs (Mitsui
et al., 2003). With these methods, it is difficult to detect the
phenotypic change caused by one of these ES cells following genetic manipulation.
As a result, previous approaches using gene-trap to identify genes involved
in ESC differentiation required the isolation of genetically modified individual
clones and the differentiation had to be handled separately (Mitsui
et al., 2003).
Furthermore, the differentiation stage of individual EBs in the same preparation
varies dramatically due to the varying size of the initial aggregate formed
and subsequent processing. Although single ESC -derived Ebs can be generated
in methylcellulose-based semisolid media (available from StemCell Technologies,
Vancouver, BC, Canada), the EB yield is low (50-100 EBs/35-mm dish) and takes
up to 20 days for differentiation (Niimi et al.,
2005). To speed up large-scale gene discovery during ES cell differentiation,
there is a need to develop a method for the differentiation of EBs from single
ESC without the requirement of isolating cells.
ALKALINE PHOSPHATASE AND ESC-SPECIFIC MARKER STAINING
Alkaline phosphatase synthesis is specific for ESC. Alkaline phosphatase (ALPL)
was detected histochemically following fixation of ES cells with 4% paraformaldehyde
and nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate toluidine
(NBT/BCIP) being used as substrate. The ALPL-expressing cells would stain dark
blue. The ESCs can be immunohistochemically detected with monoclonal antibodies
against mouse stage-specific embryonic antigen (SSEA) 1 (1:30), (SSEA) 4 (1:30),
tumor rejection antigen gp96 (TRA1; two different antibodies used for TRA1,
one recognizing a sialidase-sensitive epitope and one that reacts with an unknown
epitope; 1:20), or rabbit OCT4 (1:500). The appropriate secondary antibodies,
horse antimouse immunoglobulin G (IgG), goat anti-mouse IgM, or sheep anti-rabbit
IgG, were used to amplify the signals. Detection of specific binding was performed
with an Elite ABC peroxidase staining kit (Vector Laboratories, Inc., Burlingame,
CA) and with 3,3 9-diaminobenzidine (Vector Laboratories) as substrate. Positive
staining was gray-black in color. Staining controls using secondary antibodies
alone also were included. The putative ES cells lines were at passages 1416
at the time that marker expression was analyzed. The IVF blastocysts also were
stained as described above. The primary antibodies for SSEA1, SSEA4 and OCT4
were localized by fluorescein isothiocyanate-conjugated goat anti-mouse IgM,
horse anti-mouse IgG, or sheep anti-rabbit IgG (1:200). Finally, the samples
were washed, mounted on glass slides and examined by fluorescence microscopy
(Wang et al., 2005).
REGULATION OF DIFFERENTIATION
Figure 6A-F depict the ESCs derived embryoid
bodies. For differentiation, ESCs were washed with 10 mL Phosphate-Buffered
Saline (PBS) and then trypsinized. Well-dissociated single ESCs were transferred
to ultra low cluster 6-well plate (Corning, Corning, NY, USA) at 1000-1500 cells/well
in 1.5 mL LIF-free KnockOut DMEM supplemented with 20% FBS, pyruvate, nonessential
amino acid and mercaptoethanol.
||ESC derived embryoid bodies. Fluorescent images of embryoid
bodies (EBs) differentiated with a plate shaker--Ordinary and green fluorescent
protein-positive (GFP) embryonic stem (ES) cells were mixed at
the ratio of 3:1 before differentiation. The EBs were excited at 480 nm.
(AC) 1000 mixed cells/well in 6-well plate on day 3, day 4 (10x) and
day 5 (20x), respectively. (D) 2000 mixed cells/well on day 5 (10x). (E
and F) GFP- and GFP single ES cell-derived EBs on
day 7 after attachment to gelatin-coated dish (5x)
||Embryoid Bodies (EBs) differentiated with a plate shaker and
a spinner flask (A and B) EBs after attachment to gelatin-coated dish. (C)
Vascular-like structure in EBs shown by immunochemical staining with anti-PECAM
immunoglobulin G (IgG) (20x)
To prevent any ES cells from aggregating during the differentiation, the plate
was placed on a titer plate shaker (Lab-Line Instruments, Melrose Park, IL,
USA) at the speed of approximately 120 rpm in a cell culture incubator for 3
days. About 250 cystic EBs were either transferred onto 100 mm 0.2% gelatin-coated
dishes for attachment and further differentiation and growth or transferred
into a spinner flask. To calculate the cell numbers and shaking speed, three-fourths
of the ES cells were mixed with one-fourth of the ES cells that were constitutively
expressing green fluorescence protein (GFP) driven by an EF-1α promoter.
The cells were seeded at different densities (250-500, 500-1000, 1000-1500 and
1500-2000) and the plate was shaken at variable speeds. Only in the case where
EBs were derived from single ES cells, can the whole EB be green (GFP)
or not green at all. However, if EBs were derived from multiple ES cell aggregates,
the EB would be partially green. As shown in Fig. 6,
A, B, C and F, only
at the cell density and shaking speed defined above were the whole green and
non-green EBs obtained. Partially green EBs were seen if the cell density was
higher and the shaking speed was too low. Although single ESC -derived EBs were
obtained from the lower cell density, the size of the EB was generally smaller.
Figure 7a-c describes the differentiation
potential of embryoid bodies (Niimi et al., 2005).
Ordinary and green fluorescent protein-positive (GFP) Embryonic Stem (ES) cells were mixed at the ratio of 3:1 before differentiation.
DIFFERENTIATION OF BESC IN VIVO AND EX VIVO
The differentiation ability of bESC in vitro is evaluated by EB formation.
The EBs are aggregates of stem cells whose development is reminiscent of early
embryogenesis. Maintenance of bESC in a suspension culture (Strelchenko
1996) or in the absence of a feeder layer (Saito et
al., 2003; Wang et al., 2005) initiates
the formation of EBs. The EBs are composed of two layers of cells, ectoderm-like
cells covered by a thin layer of endoderm like cells, with heterogeneous cellular
particles within the cavity. The cells of bovine EBs give rise to a wide variety
of differentiated cell types, including derivatives of the three germ layers
(Saito et al., 2003; Wang
et al., 2005). This ability is a proof of their pluripotent-differentiation
character in vitro. The ability of bESC to participate in the embryogenesis
has been proven only after a short propagation period in vitro. Chimeric
transgenic calves have been born after injection of bESC (passage 3) into cleavage
stage embryos (Cibelli et al., 1998), embryo
aggregation with bESC (passage 9 -13) (Iwasaki et al.,
2000). Yet integration of ESCs into the germ line, one of the properties
used to define ESCs, have not been achieved in any of these experiments. Calves
were also successfully cloned using ES-like cells as donor nuclei (short cultured
ICMs or passage 14-18) (Sims and First 1994; Saito
et al., 2003), but the use of ESC in nuclear transfer cannot be taken
as a proof of pluripotency as cloned animals have been produced from fully differentiated
somatic cell nuclei (Kato et al., 2000; Wakayama
and Yanagimachi, 2001). Figure 8 describes the isolation
and identification of mouse embryonic stem cells.
|| Isolation and Identification of mouse embryonic stem cells
APPLICATIONS OF ESC
Figure 9 depicts the salient applications of ESC. The potential
application of ESCs depends on the derivation of specialized lines of differentiated
cells. But in real sense these are challenges which needs to be prevailed
for the successful application of ESCs. For these reasons, three methods of
therapeutic cloning were proposed, suitable for preparing pluripotent human
embryonic stem cells with well defined genetic information from which desired
differentiation would then follow:
||The replacement of the nucleus of an oocyte with the nucleus
of an adult cell of a given subject, followed by embryonic development to
the stage of blastocyst and the use of the Inner Cell Mass (ICM) in order
to obtain ES cells and, from these, the desired differentiated cells
||The transfer of a nucleus of a cell of a given subject into
an oocyte of another animal. An eventual success in this procedure should
lead to the development of a human embryo, to be used as in the preceding
||The reprogramming of the nucleus of a cell of a given subject by fusing
the ES cytoplast with a somatic cell karyoplast, thus obtaining a cybrid.
This is a possibility which is still under study. In any event, this method
too would seem to demand a prior preparation of ES cells from human embryos.
Figure 8 describes the salient applications of ESCs
|| Application of ESC in therapeutics and advances in developmental
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