Báo cáo khoa học: "Human embryonic stem cells and therapeutic cloning" pdf

JOURNAL OF
Veterinary
Science
J. Vet. Sci. (2005), 6(2), 87–96
Human embryonic stem cells and therapeutic cloning
Woo Suk Hwang *, Byeong Chun Lee , Chang Kyu Lee , Sung Keun Kang
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
The Xenotransplantation Research Center, Seoul National University Hospital, Seoul 110-744, Korea
School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea
The remarkable potential of embryonic stem (ES) cells
is their ability to develop into many different cell types. ES
cells make it possible to treat patients by transplanting
specialized healthy cells derived from them to repair
damaged and diseased cells or tissues, known as “stem cell
therapy”. However, the issue of immunocompatibility is
one of considerable significance in ES cell transplantation.
One approach to overcome transplant rejection of human
ES (hES) cells is to derive hES cells from nuclear transfer
of the patient’s own cells. This concept is known as
“therapeutic cloning”. In this review, we describe the
derivations of ES cells and cloned ES cells by somatic cell
nuclear transfer, and their potential applications in
transplantation medicine.
Key words: embryonic stem cell, somatic cell nuclear transfer,
stem cell, pluripotency
Introduction
Stem cells can replicate themselves and generate into
more specialized cell types as they multiply. There are two
kinds of stem cells in the body, originated from embryonic
or adult tissues. Adult stem cells are undifferentiated cells
found among differentiated cells in a tissue or organ. They

can renew themselves, and can differentiate to yield the
major specialized cell types of the tissue or organ.
Embryonic stem (ES) cells are derived from a blastocyst that
is developed from in vitro fertilized egg. The remarkable
potential of stem cells is their ability to develop into many
different cell types, which serves as a sort of repair system
for the body. Stem cells make it possible to treat patients by
transplanting specialized healthy cells produced from them
to repair damaged and diseased body-parts. This concept is
known as “stem cell therapy” [37]. Stem cell therapy is now
emerging as a potentially revolutionary new way to treat
disease and injury, with wide-ranging medical benefits.
Stem cell therapy has potential applications in treating a
wide array of diseases and ailments of the brain, internal
organs, bone and many other tissues. Such ailments include
strokes, Alzheimer’s and Parkinson’s diseases, heart disease,
osteoporosis, insulin-dependent diabetes, leukemia, burns
and spinal-cord injury. Both adult and ES cells can be used
for stem cell therapy. In this review, we describe the
derivation and characterization of ES cells and cloned ES
cells. Furthermore, current perspectives of potential
applications of stem cells for tissue repair and
transplantation medicine are also reviewed.
Derivation and culture of ES cells
In the 1980’s, ES cells were first established from
preimplantation murine embryos [19,42]. Mouse ES cells
were derived from the inner cell mass (ICM) of an expanded
blastocyst at 3.5 days post-coitum or from delayed blastocysts
collected at 4-6 days after ovariectomy. Interestingly, mouse
ES cells were isolated only from permissive strains of mice,

129/SV or 129/Ola, to obtain totipotent cells [63,49,52]. For
establishing ES cells, ICM is isolated by immunosurgery to
remove trophoblast cells. After several days in culture,
isolated ICM cells form a colony that can be expanded by
disaggregating and re-seeding on non-proliferative mitomycin-
C treated or irradiated fibroblasts (STO cells or primary
mouse embryonic fibroblasts) [1,27,63]. In order to prevent
spontaneous differentiation, ES cells must be maintained by
repeated passages on feeder layers, usually a feeder layer is
generally required to isolate ES cells and to support their
successive passages [74]. The main role of feeder cells is
probably to provide growth factors necessary for proliferation
and inhibition of spontaneous differentiation. The principal
differentiation inhibitory factor is leukemia inhibitory factor
(LIF), as demonstrated that LIF-defective fibroblasts cannot
maintain ES cells as undifferentiated state [72], and LIF in
the medium can support ES cells without feeder cells
[52,74]. LIF is a pleitrophic cytokine that acts through the
gp130 pathway [86], which is common to related cytokines
such as ciliary neurotrophic factor [13], oncostatin M [64],
*Corresponding author
Tel: +82-2-880-1280, Fax: +82-2-884-1902
E-mail:
Review
88 Woo Suk Hwang et al.
and interleukin-6 [48]. Each of these cytokines can maintain
the pluripotency of ES cells. Standard culture conditions for
ES cells contain fetal bovine serum (FBS), which is not well
characterized and is susceptible to variation from batch to
batch. ES cells can also be maintained less effectively

without feeder layer on gelatin or extracellular matrix
substrate in conditioned medium or in LIF-supplemented
medium [80].
In addition to mouse ES cells, isolation of ES cells have
been attempted rats [30], mink [75], rabbits [22], hamster
[15,56], primates [78], sheep [55,25], cattle [20,73], and
pigs [55,50,21,76,45]. A wide range of pluripotency has
been demonstrated in ES cells from each species, but only in
the mouse, germline chimeras were produced [62]. Porcine
ES-like cells were derived from early pig embryos, but lost
their pluripotency over time in culture [45]. Although
chimeras were produced from freshly isolated porcine ICMs
injected into host blastocysts, the ability of chimera
production was lost after culturing porcine ICM in vitro [5].
This may be due to improper culture conditions and/or a
requirement for species-specific growth factors. Further
improvements in culture conditions are required to isolate
pluripotent stem cells from pigs. Therefore, despite extensive
research efforts, no proven ES cells with satisfying all
criteria to be a pluripotent cells were established in any
species other than the mouse [39].
In 1998, human embryonic stem (hES) cells were first
isolated from in vitro fertilized blastrocysts [77], using mouse
embryonic fibroblasts as feeder cells and serum-containing
medium. Human ES cells are typically cultured with animal-
derived serum or serum replacement on mouse feeder
layers. It was demonstrated that culturing human ES cells
with serum replacement on mouse feeder cells are the
sources of the nonhuman sialic acid Neu5Gc, which could
induce an immune response upon transplantation of hES

cells into patients [43]. Many efforts have been recently
made to eliminate these animal-derived components and to
culture hES cells on feeder-free conditions or human feeder
cells for safe transplantation of human ES cells. The use of
feeder-free systems, such as Matrigel or other components
of the extracellualr matrics, have been explored [83,17,
65,4]. However, matrix components used for feeder-free
culture are still from animal sources and the medium also
contains animal-derived products. Human feeders of
different origin have also been tried and support the growth
of hES cells [2,3,10,28,44,59,60]. With much progress in
research on hES cell culture, the safe standard culture
condition for hES is expected to be established for
transplantation of hES cells into patients. As of 2003, 71
independent hES cell lines identified worldwide. Among
them, 11 cell lines are currently available for research
purposes with limited published data on their culture and
differentiation characteristics [87]. Recently, more hES cells
are being established and the numbers are growing abruptly
[14]. A breakthrough in hES cell research was reported in
2004, i.e. derivation of immune-compromised hES cells
using somatic cell nuclear transfer (SCNT) [29].
Characteristics of ES cells
ES cells show a high nucleo-cytoplasmic ratio and large
nucleoli, indicating active transcription and a correlative
high protein synthesis at least relevant to active cell
proliferation. ES cells express cell markers that can be used
to characterize undifferentiated ES cells. A common marker
for the undifferentiated state is alkaline phosphatase [82]
which is equivalent to non-specific alkaline phosphatase of

the ICM of the mouse blastocyst. Other undifferentiated
markers generally correspond to carbohydrate residues of
membrane proteins including ECMA-7 [36] and SSEA-1
[69]. The germline specific transcription factor, Oct-4, is
also a reliable marker for undifferentiated embryonic cells
and ES cells [54]. Each of these markers is down-regulated
upon differentiation of ES cells.
Because ES cells are pluripotent under specific conditions,
they are differentiated into cells of multiple lineages in vitro
[51]. The conditions required to induce differentiation
include a high number of passages, absence of LIF and/or
feeder cells, or the addition of differentiation factors such as
retinoic acid (RA) or dimethyl sulfoxide. When ES cells are
cultured at high cell density on a non-adhesive surface, they
form round embryoid bodies showing many similarities to
embryo development in vivo [16]. The embryoid bodies
develop an outer layer of endoderm-like cells and eventually
a central cavity, resulting in a cystic embryoid body. When
these cells are allowed to attach again and form outgrowths,
embryoid bodies can give rise to differentiated tissues such
as myocardium, blood islands and hematopoietic stem cells
[16,51]. ES cells can also be differentiated in in vivo. When
ES cells or embryoid bodies are implanted into immunodeficient
mice, highly differentiated tissues can be obtained [9]. More
importantly, when injected into a morula or into the cavity of
an expanded blastocyst, ES cells give rise to chimeric mice
in which ES cells take part in the development of all types of
tissue including the germ line [62].
Applications of stem cells for tissue repair and
transplantation medicine

There are several approaches in human clinical trails that
employ adult stem cells (such as blood-forming hematopoietic
stem cells and cartilage-forming cells). A potential
advantage of using adult stem cells is that the patient's own
cells could be expanded in culture and then reintroduced
into the patient without immune rejection. However,
because adult cells are already specialized, their potential to
regenerate damaged tissue is limited. Another limitation of
adult stem cells is their inability to effectively grow in
Human embryonic stem cells and therapeutic cloning 89
culture. Therefore, obtaining clinically significant amounts
of adult stem cells may prove to be difficult. In contrast, ES
cells can become any and all cell types of the body and large
numbers of ES cells can be relatively easily obtained in vitro
culture. Therefore, ES cells could be the choice of cells in
stem cell therapy for various diseases. One of the critical
steps for stem cell therapy using ES cells is to produce
desired type of cells by differentiation. As mouse ES cells,
hES cells can form embryoid body in suspension culture,
which is the typical structure of spontaneously differentiated
ES cells compromising all three germ layers in vitro [2].
Treating embryoid bodies with growth factors or differentiation
inducing agents such as fibroblasts growth factor (FGF)-2 or
RA influences the outcome of differentiation [66]. These
approaches of differentiation are widely used in isolating
and analyzing lineage-specific human precursor cells from
ES cell cultures. In addition to spontaneous differentiation,
many researches have attempted to control the differentiation
of ES cells. Either supplementing culture media with growth
factors or co-culturing ES cells with the inducing cells

induced differentiation of a specific lineage or increased
population of specific cells during spontaneous differentiation
[53]. Human ES cells have shown to be differentiated into
the various cell types from each of three germ layers in a
controlled manner. These include ectodermal origin;
neuronal cells, keratinocytes or adrenal cells [8,58,88,23],
mesodermal origin; hematopoietic precursors, endothelial,
cardiomyocyte or osteocyte [32,34,35,41,84,46,70], and
endodermal origin; pancreatic cells or heparocytes [66,6,57].
Furthermore, hES cells, unlikely murine ES cells, can
differentiate into trophoblast cells or extraembryonic
endoderm [77,85], representing a useful model for studying
human placental development and function. Although
numerous key factor(s) or step(s) for guided differentiation
have been presented, the nature of complex culture system
makes it impossible to delineate precise pathway for specific
cell differentiation. Therefore, optimization of current
protocols and/or development of novel methods for
precisely controlled differentiation of hES cells are crucial
to facilitate the application of hES cells into clinical stem
cell therapy.
Production of immunocompatible cloned ES
cells by somatic cell nuclear transfer in animals
For transplantation of ES cells, the issue of
immunocompatibility is one of considerable significance. If
the transplanted cells are grown from stem cells that are not
genetically compatible with a patient, their immune system
will reject the cells. It has been proposed that in vitro
fertilized hES cells could be transplanted back to the patients
to cure numerous diseases without immune rejection.

However, this hypothesis was rejected because it was
demonstrated that while undifferentiated hES cells express
only low levels of major histocompatibility complex 1
(MHC-1) molecules which activate an immune response,
hES cells upon differentiation express the molecules,
indicating that immune rejection can be occurred [18]. The
strategy being proposed for immunocompatibility of stem
cell transplantation is the creation of hES cell bank that will
accommodate all different immune types of hES cells for all
potential patients. However, it will need to huge number of
hES cells to match with all type of histocompatiblity
complex. The isolation of pluripotent hES cells [77] and
breakthroughs in somatic cell nuclear transfer (SCNT) in
mammals [81] have raised the possibility of performing
human SCNT to generate virtually unlimited sources of
undifferentiated cells, with potential applications in tissue
repair and transplantation medicine. This concept, known as
“therapeutic cloning”, is suggested as an alternate potential
way of avoiding immune problems because it will generate
isogenic or ‘tailor-made’ hES cells which all nuclear genes
would be recognised as from the same origin [37,26,31].
Therapeutic cloning refers to the transfer of the nucleus of a
somatic cell into an enucleated donor oocyte. In theory, the
oocyte’s cytoplasm would reprogram the transferred nucleus
by silencing all the somatic cell genes and activating the
embryonic ones. The reconstructed embryos are induced
embryonic developments and ES cells would be isolated
from the ICMs of the cloned preimplantation embryo. When
applied in a therapeutic setting, these cells would carry the
nuclear genome of the patient; therefore, it is proposed that

following directed cell differentiation, the cells could be
transplanted without immune rejection for treatment of
degenerative disorders such as diabetes, osteoarthritis, and
Parkinson’s disease, among others.
The idea of reactivating embryonic cells in somatic cells
by nuclear transplantation was first put forward by Spemann
in 1914’s using newt eggs [71]. This concept was later
applied to more terminally differentiated cells in amphibian
by Gurdon et al. [24], culminating with the currently
accepted idea that mammalian somatic cells can be turn into
a whole new individual when placed in the egg of the same
species. In an attempt to generate embryonic cells from
somatic cells, in 1998 Dr. Cibelli et al. performed nuclear
transfer of bovine fibroblasts into enucleated bovine oocytes
[11]. They generated thirty seven cloned blastocysts from
330 reconstructed eggs and isolated 22 ES-like cell lines
from them. When these ES-like cells are injected into host
non-transgenic bovine embryos, 6 out of seven calves were
found to have at least one transgenic tissue in them.
Subsequently in 2000, Munsie et al. [47] and Kawase et al.
[33] showed similar results using mouse cumulus cells as
nuclear donors and demonstrated that these mouse nuclear
transfer-derived ES cells were capable of in vitro
differentiation [47]. In 2001, Wakayama et al. demonstrated
that dedifferentiated cloned mouse ES cells derived from
nuclear transfer of cumulus cells can go to the germline and
90 Woo Suk Hwang et al.
produce offspring [79]. The same group also demonstrated
that neurons derived from somatic-cell-cloned-ES cells can
produce dopamine and serotonin [7]. In 2002, Rideout et al.

showed that somatic cells isolated from a Rag (-) mouse, i.e.
an animal that lacks T and B cells, can be transformed into
ES cells genetically corrected for the Rag mutation and then
turned into blood progenitors that will generate B and T
cells when reintroduced into the mutant animal [61]. This
experiment demonstrated that SCNT can be used as a reliable
tool for ex-vivo gene therapy. Furthermore, transplantation
of cloned mouse ES cells derived from SCNT [79] has been
successfully applied to treat Parkinson’s disease in Parkinsonian
mice [7].
Establishment and characterization of human
cloned ES cells
Having thoroughly the proved concept of therapeutic
cloning in animals, we set up to test whether the SCNT for
the purpose of making ES cells was feasible in man.
Recently, Cibelli et al. [12] demonstrated the development
of cloned human embryos to 8 to 10 cell stages, but failed
obtained blastocysts for derivation of human cloned ES
cells. Therefore, no information or protocols for obtaining
human cloned blastocysts were available. Absent any report
of an efficient protocol for human SCNT, several critical
factors are needed to be determined and optimized. Our
experiences with domestic animals indicate that reprogramming
time, oocyte activation method and in vitro culture
conditions play a critical role on chromatin remodelling and
the developmental competence of SCNT embryos. These
three critical factors were optimized throughout the
experiments (Table 1). First, the reprogramming time
defined as the period of time between cell fusion and oocyte
activation is needed to return the gene expression pattern of

the somatic cell to one that is appropriate and necessary for
the development of the embryo. In our study, by allowing
two hours for reprogramming to allow proper embryonic
development, we were able to obtain ~25% of human
reconstructed embryos to develop to the blastocysts (Table
1). Second, oocyte activation is naturally the role of the
sperm. During fertilization, the spermatozoa will trigger
transient calcium release inside the oocytes of a particular
magnitude and frequency that lead to a cascade of events
culminating with first embryonic cell division. Since sperm-
mediated activation is absent in SCNT, an artificial stimulus
is needed to initiate embryo development. We found that
10 mM ionophore for 5 min followed by incubation with
2.0 mM 6-dimethyl amino purine had proven to be the most
efficient chemical activation protocol for human SCNT
embryos (Table 1). Third, in order to overcome inefficiencies
in embryo culture, we prepared the human modified
synthetic oviductal fluid (SOF) with amino acids
(hmSOFaa) by supplementing mSOFaa with human serum
Table 1 . Conditions for human somatic cell nuclear transfer
Experiment Activation condition*
Reprogramming

time (hrs)
1 step
medium
2 step
medium
No. of
oocytes

No. (%) of cloned embryos
developed to
2-cell
Compacted
morula
Blastocyst
1
set
10 µM Ionophore 6-DMAP 2 G 1.2 hmSOFaa 16 16 (100) 4 (25) 4 (25)
10 µM Ionophore 6-DMAP 4 G 1.2 hmSOFaa 16 15 (94) 1 (6) 0
10 µM Ionophore 6-DMAP 6 G 1.2 hmSOFaa 16 15 (94) 1 (6) 1 (6)
10 µM Ionophore 6-DMAP 20 G 1.2 hmSOFaa 16 9 (56) 1 (6) 0
2
set
10 µM Ionophore 6-DMAP 2 G 1.2 hmSOFaa 16 16 (100)
5 (31) 3 (19)
5 µM Ionophore 6-DMAP 2 G 1.2 hmSOFaa 16 11 (69)
00
10 µM Ionomycin 6-DMAP 2 G 1.2 hmSOFaa 16 12 (75)
00
5 µM Ionomycin 6-DMAP 2 G 1.2 hmSOFaa 16 9 (56)
00
3 set
10 µM Ionophore 6-DMAP 2 G 1.2 hmSOFaa 16 16 (100)
4 (25) 3 (19)
10 µM Ionophore 6-DMAP 2 G 1.2 G 2.2 16 16 (100)
00
10 µM Ionophore 6-DMAP 2 Continuous hmSOFaa 16 16 (100)
00
4 set 10 µM Ionophore 6-DMAP 2 G 1.2 hmSOFaa 66 62 (93)

24 (36) 19 (29)
*Fused donor oocytes and somatic cells were activated in either calcium ionophore A23187 (5 or 10 µM) or ionomycin (5 or 10 µM) for 5 min,
followed by 2 mM 6-dimethylaminopurine (DMAP) treatment for 4 hrs.
Oocytes were incubated in first medium for 48 hrs.
Human embryonic stem cells and therapeutic cloning 91
albumin and fructose instead of bovine serum albumin and
glucose, respectively. We observed that culturing human
SCNT-derived embryos in G1.2 medium for the first 48 hrs
followed by hmSOFaa medium produced more blastocysts,
compared to G1.2 medium for the first 48 hrs followed by
culture in G1.2 medium or in continuous hmSOFaa medium
(Table 1). The protocol described here produced cloned
blastocysts at rates of 19 to 29% and was comparable to
those from established SCNT methods in cattle (~25%) and
pigs (~26%).
As results, the reconstructed oocytes were developed to 2-,
4-, 8 to 16-cell, morulae and blastocysts (Fig. 1A to F). A
total of 30 SCNT-derived blastocysts (Fig. 1F) after removal
of zona pellucida with 0.1% pronase treatment were
cultured, 20 ICMs were isolated by immunosurgical
removal of the trophoblast (Fig. 2A), first incubating them
with 100% anti-human serum antibody for 20 min, followed
by an additional 30 min exposure to guinea pig complement.
Isolated ICMs were cultured on mitomycin C mitotically
inactivated primary mouse embryonic fibroblast feeder
layers in gelatin-coated 4-well tissue culture dishes. The
culture medium was Dulbecco’s modified Eagle’s Medium
(DMEM)/DMEM F12 (1 : 1) supplemented with 20%
Knockout Serum Replacement, 0.1 mM β-mercaptoethanol,
1% nonessential amino acids, 100 units/ml penicillin, 100

µg/ml streptomycin, and 4 ng/ml basic fibroblast growth
factor (bFGF). During the early stage of SCNT ES cell
culture, the culture medium was supplemented with
2,000 units/ml human leukaemia inhibitory factor (LIF). As
results, one ES cell line (SCNT-hES-1) was derived. The
cell colonies display similar morphology to that reported
previously for hES cells derived from IVF (Fig. 2B and C).
The SCNT-hES-1 cells had a high nucleus to cytoplasm
ratio and prominent nucleoli (Fig. 1F). When cultured in the
defined medium conditioned for neural cell differentiation
[40], SCNT-hES-1 cells differentiated into nestin positive
cells, an indication of primitive neuroectoderm differentiation.
The SCNT-hES-1 cell line was mechanically passaged
every five to seven days using a hooked needle and
successfully maintained its undifferentiated morphology
after continuous proliferation for >140 passages, while still
maintaining a normal female (XX) karyotype. When
characterized for cell surface markers, SCNT-hES-1 cells
express ES cell markers such as alkaline phosphatase,
Fig. 1. Preimplantation development of embryos after somatic cell nuclear transfer (SCNT). The fused SCNT embryo (A) was
developed into 2-cell (B), 4-cell (C), 8-cell (D), morula (E) and blastocyst (F). ×200 (A to E) and ×100 (F). Scale bar; 100 µm (A to E)
and 50 µm (F).
Fig. 2. Morphology of inner cell masses (ICMs) isolated from cloned blastocysts (A, ×100) by immunosurgery and the phase contras
t
micrographs of a colony of SCNT-hES-1 cells (B, ×100), and higher magnification (C, ×200). Scale bar; 50 µm (A) and 100 µm (B an
d
C).
92 Woo Suk Hwang et al.
SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4, but not
SSEA-1 (Fig. 3). As previously described in monkey [78]

and human ES cells [77,58], and mouse SCNT-ES cells
[33], SCNT-hES-1 cells do not respond to exogenous
leukaemia inhibitory factor (LIF), suggesting that a
pluripotent state is maintained by a gp130 independent
pathway. Pluripotency of SCNT-hES-1 cells was tested in
vitro and in vivo. For embryoid body formation, clumps of
the cells were cultured in vitro for 14 days in suspension
using plastic Petri dishes in DMEM/DMEM F12 without
hLIF and bFGF. The resulting embryoid bodies were stained
with three dermal markers and were found to differentiate
into a variety of cell types including derivatives of
endoderm, mesoderm, and ectoderm. When undifferentiated
SCNT-hES-1 cells (clumps consisting of about 100 cells)
were injected into the testis of six- to eight-week-old SCID
mice, teratomas were obtained from six to seven weeks after
injection. The resulting teratomas contained tissue representative
of all three germ layers including neuroepithelial rosset,
pigmented retinal epithelium, smooth muscle, bone,
cartilage, connective tissues, and glandular epithelium. In
order to confirm SCNT-origin of our cells, not from the
pathenogenetic activation of oocyte, the DNA fingerprinting
analysis with human short tandem repeat (STR) markers
was performed and demonstrated that the cell line originated
from the cloned blastocysts reconstructed from the donor
cells, not from parthenogenetic activation. The statistical
probability that the cells may have derived from an
unrelated donor is 8.8 × 10
. Furthermore, the RT-PCR
amplification for paternally-expressed (hSNRPN and ARH1)
and maternally-expressed (UBE3A and H19) genes

demonstrated biparental, and not unimaternal, expression of
imprinted genes. Confirmation of complete removal of
oocyte DNA, DNA fingerprint assay and imprinted gene
analysis provide three lines of evidence supporting the
SCNT origins of SCNT-hES-1 cells.
Discussion and Conclusion
Success in the production of human SCNT-ES-1 cell line
was attributed to optimization of several factors including
the donor cell type, reprogramming time, activation protocol
and use of sequential culture system with newly developed
in vitro culture medium as described above. Furthermore,
use of less-invasive enucleation method (a squeezing
method) is suggested to be one of key factor. The MII
oocytes were squeezed using a glass pipet so that the DNA-
spindle complex is extruded through a small hole in the zona
pellucida, instead of aspirating the DNA-spindle complex
with a glass pipette as others have described [81]. With use
of an aspiration method, Simerly et al. [66] failed to
obtained monkey cloned blastocysts because of defective
mitotic spindles after SCNT in non-human primate embryos,
perhaps resulting from the depletion of microtubule motor
and centrosome proteins lost to the meiotic spindle after
enucleation. However, using a squeezing method, they are
Fig. 3. Expression of characteristic cell surface markers in human SCNT ES cells. SCNT-hES-1 cells expressed cell surface markers
including alkaline phosphatase (A), SSEA-3 (C), SSEA-4 (D), TRA-1-60 (E), TRA-1-81 (F), and Oct-4 (G), but not SSEA-1 (B). ×40.
Scale bar; 100 µm.
Human embryonic stem cells and therapeutic cloning 93
successful in obtaining monkey cloned blastocysts [67],
supporting our idea that use of a squeezing method is
attributed to obtaining human cloned blastocysts.

In order to successfully derive immunocompatible human
ES cells from a living donor, a reliable and efficient method
for producing cloned embryos and ES isolation must be
developed. Thomson et al. [77], Reubinoff et al. [58], and
Lanzendorf et al. [38] produced human ES cell lines at high
efficiency. Briefly, five ES cell lines were derived from a
total of 14 ICMs, two ES cell lines from four ICMs, and
three ES cell lines from 18 ICMS, respectively. In our study,
one SCNT-hES cell line was derived from 20 ICMs. It
remains to be determined if this low efficiency is due to
faulty reprogramming of the somatic cells or subtle
variations in our experimental procedures. We cannot rule
out the possibility that the genetic background of the cell
donor had an impact on the overall efficiency of the
procedure. Further improvements in in vitro culture system
for ES cells are needed before contemplating the use of this
technique for cell therapy. In addition, those mechanisms
governing the differentiation of human tissues must be
elucidated in order to produce tissue-specific cell populations
from undifferentiated ES cells. In conclusion, our study
describes the first establishment of pluripotent ES cells from
SCNT of a human adult reprogrammed cell and provides the
feasibility of using autologous cells in transplant medicine.
With this approach for overcoming transplant rejection, ES
cells will provide a promising potential to treat a variety of
degenerative diseases.
Acknowledgments
This study was supported by grants (Biodiscovery Program)
from the Ministry of Science and Technology, Korea.
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