J O U R N A L O F

Veterinary Science

J. Vet. Sci. (2005), 6(2), 87–96

Human embryonic stem cells and therapeutic cloning

Woo Suk Hwang1,2,3,

*, Byeong Chun Lee1,2

, Chang Kyu Lee3, Sung Keun Kang

1,2

1Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea2The Xenotransplantation Research Center, Seoul National University Hospital, Seoul 110-744, Korea3School 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 authorTel: +82-2-880-1280, Fax: +82-2-884-1902E-mail: hwangws@snu.ac.kr

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 andtransplantation 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 EScells 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 humancloned 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)1st step

medium†

2nd step medium

No. of oocytes

No. (%) of cloned embryos developed to

2-cellCompacted

morulaBlastocyst

1st 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

2nd 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) 0 0

10 µM Ionomycin 6-DMAP 2 G 1.2 hmSOFaa 16 12 (75) 0 0

5 µM Ionomycin 6-DMAP 2 G 1.2 hmSOFaa 16 9 (56) 0 0

3rd 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) 0 0

10 µM Ionophore 6-DMAP 2 Continuous hmSOFaa 16 16 (100) 0 0

4th 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) wasdeveloped 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 contrastmicrographs of a colony of SCNT-hES-1 cells (B, ×100), and higher magnification (C, ×200). Scale bar; 50 µm (A) and 100 µm (B andC).

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 × 10−16. 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 markersincluding 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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