Human Embryonic Stem Cell Research :Today and Tomorrow
Shin-Yong Moon M.D. Ph.D.
Department of Obstetrics and Gynecology
College of Medicine
Seoul National Universisty
#28 Yungun-Dong, Chongno-Gu, Seoul, 110-744, Korea
PART I. Human Embryonic Stem Cells: The Korean Experience
Embryonic stem (ES) cell research has a history of more than 20 years, and has made some outstanding
contributions to our understanding of mouse embryology. ES cell, which is derived from inner cell mass
(ICM) in preimplantation embryos, could proliferate without limitation in vitro and have pluripotentency.
Stem cells differ from other kind of cells in the body. All stem cells – regardless of their source – have
three general properties: they are capable of dividing and renewing themselves for long periods; they are
unspecialized; they can give rise to specialized cell types. These cells could differentiate into derivatives
of three embryonic germ layers including ectoderm, mesoderm and endoderm, in proper culture
conditions. According to these characteristics of human ES cells, many researchers are trying to adapt
stem cell to many biological and medical fields. Stem cell can be used as a source for study of basic
developmental biology, identification of factors that involved in regulation of developmental process and
differentiation into certain cell or tissue, screening for drugs and toxins, and transplantation therapy.
Thomson et al and Reubinof et al reported the derivation of ES cell lines from human blastocyst. We
confirm these results and observe the characterization of the human ES cell. We described that the
establishment of three human embryonic stem cell lines, SNUhES-1,-2 and -3 from frozen 2 pronuclear
Donated Embryos for Establishment of ES Cell lines
ES cells were derived from cryopreserved 2 pronuclear stage embryos that have been fertilized in vitro —
in an in vitro fertilization (IVF) clinic of Seoul National University Hospital — and then donated for
research purposes with informed consent of the donors and Institutional Review Board approval.
Seventy-three frozen-thawed donated 2 pronuclear stage embryos produced by IVF were cultured to the
blastocyst stage in G1.2 and G2.2 medium. Ten blastocysts that developed were selected for ICM
Culture Media and Preparation of STO Feeder Layer
Media for ES culture consisted of knockout DMEM(KO-DMEM) or DMEM/F12 supplemented with
20% knockout serum replacement, 2mM L-glutamine, 1% nonessential amino acid, 0.1mM β-
mercaptoethanol, 0.5% penicillin-streptomycin . Prepared culture media were incubated in 37℃, 5%
CO2 incubator for 24hours before using..
STO Feeder Layer
Frozen STO mouse fibroblast (ATCC,USA) were thawed and cultured for 3days. Culture medium
consisted of DMEM (high glucose, with L-glutamine) supplemented with 10% FBS, 1mM sodium
pyruvate, 1.5g/L sodium bicarbonate,and 0.5% penicillin-streptomycin. Outgrowthed STO fibroblast
layers were treated with trypsin-EDTA for dissociation and harvested. Collected STO mouse fibroblasts
were treated with mitomycin (Gibco,USA ). Frozen mitomycin treated STO cells were thawed in 37℃
and seeded on 0.1% gelatin pre-coated culture dish before using. After 24 hours, the medium was
exchanged with ES culture medium.
Isolation of Inner Cell Mass and ES Cell culture
Whole embryo culture
After digestion of zona pellucida with pronase, the embryo cultured on mitomycin treated STO feeder
layer on gelatin-coated tissue culture plate in 37℃, 5% CO2 incubator. After 48 hours of plating, culture
media were exchanged daily for half of the volume. After 7-8 days of plating, central part of the cell
except trophoblsat was isolated mechanically and replated on fresh feeder layer. Once established and
expanded, cultures were passaged by selection of individual colonies by micropipette.
Isolation inner cell mass by immunosurgery
Blastocyst was treated with pronase about 10-15 min to dissolve zona pellucida and outer
trophoecdtoderm layer was removed by the treatment of anti-human polyvalent immunoglobulins (Sigma,
USA) and guinea pig complement(Gibco,USA) to isolate inner cell masses. Isolated inner cell masses
were then plated onto a STO feeder layer.
After 48 hours of plating, culture media were exchanged daily for half of the volume. Within 7-8 days of
plating, clump of the cell was isolated mechanically and replated on fresh feeder layer.
Culture of hES cell
Expanded blastocysts from cryopreserved 2-pronuclear stage embryos were used for establishment of
human embryonic stem cell lines. Ten inner cell masses were isolated by immunosurgery or whole
embryo culture and plated onto fresh mouse STO feeder layer and three ES cell lines originating from
three separate embryos were derived. Culture media were exchanged daily for half of the volume. After
culture of 5 -7 days, groups of small, tightly packed cells had begun to proliferate from inner cell mass.
The small cells were mechanically dissoiated from outgrowths of differentiated cells, and following
replating they give rise to flat colonies of cells. ES colonies were dissociated containing 200~300 cells
by glass pipette under the stereomicroscope. Morphologies of the ES cells were observed by inverted
phase contrast microscope.
Cells in colonies of ES cells have high ratio of nucleus to cytoplasm and prominent nucleoli. Nucleolus
occupied most of the cell. They also show certain border and formed flat colonies. Because ES cell
continuously pushed out the feeder layers, the border part of the ES colonies showed high density of the
cell. Cell line SNUHES-1 has been grown for 55 passages in vitro, SNUHES-2 for 72 passages and
SNUHES-3 for 43 passages respectively.
Established undifferentiated ES cells showed spontaneous differentiation some time. Differentiation
usually started in central part of the colony and showed enlarged cytoplasm like endoderm. In vitro, the
ES cells differentiated when cultured in the absence of mouse embryonic fibroblast feeder layers. both in
the presence and absence of human inhibitory leukemia factor. Leukemia inhibiting factor (LIF) was
supplemented until 5 passages.
Characterization of Embryonic Stem Cell
The human ES cell lines expressed cell surface markers that characterize undifferentiated nonhuman
primate ES cells, including alkaline phosphatase, stage specific embryonic antigen (SSEA)-3, SSEA-4.
Marker analysis was performed on SNUHES-1 at passage levels8-10 and on SNUHHES-2 at passage
levels 6-8, and on SNUHES-3 at passage level 12-14. After then we did marker analysis every 10
Expression of Alkaline phosphatase activity
The activity of alkaline phosphatase, which is the cell surface marker of the undifferentiated cells, was
tested to confirm the characteristics of undifferentiated embryonic stem cell.
Histochemical staining was performed by alkaline phosphatase diagnostic kit (Sigma, USA). Media were
discarded and washed twice with PBS. Then fixed with citrate-acetone-formaldehyde solution for 1 min
and washed twice with PBS for 2 min. 15 min after addition of Naphthol AS-BI alkaline solution, washed
with PBS for 15 min and observed for color reaction.
All of the established cell line stained positively for alkaline phosphatase activity. But colonies in
spontaneous differentiation didn't show activity.
Stage specific embryonic antigen-1, 3 and 4.
Antibody MC 480 for SSEA-1, MC 631 for SSEA-3 and MC183-70 for SSEA-4 were used and diluted
1:100. Mouse Ig (Vectastain kit) was used as secondary antibody. Media were discarded and ES cell
were fixed with 4% paraformaldehyde for 30 min. After fixation, 3% hyperoxide solution were added and
incubated for 10 min followed by washing three times for 5min. To prevent nonspecific reaction, normal
goat serum were treated for 1hr. Primary antibodies were treated for 1hr and washed with PBS.
Secondary antibodies were treated for 45min and washed three times with PBS. ABC (avidin biotin
peroxidase complex, Vector) were treated for 30 min. finally, peroxidase substrate kit DAB (Vector) were
treated for 20 min.
Human ES cells did not express SSEA-1, a marker for mouse ES cell. Occasional colonies consisted of
non-stained, central, undifferentiated cells surrounded by a margin of stained, differentiated, epithelial
cells. Most colonies contained a mixture of weakly stained cells and a majority of non-stained cells for
SSEA-3. Staining intensity for SSEA-4 on the human ES cell lines was consistently strong, but staining
intensity for SSEA-3 was weak and varied both within and among colonies.
RT-PCR Analysis for Oct-4.
Total RNA from ES cell was extracted by RNeasy mini kit (Qiagen). Total 1㎍ of RNAs were used as a
template for first strand synthesis. First strand were incubated at 42℃ for 60min and used for second
strand synthesis. PCR were performed at 61℃ for 40 sec in 34 cycle.
β-actin(F:CGCACCCACTGGCATTGTCAT, R:TTCTCCTTGATGTCACGCAC) and Oct-4
(F:GGCGTTCTCTTTGGAAAGGTGTTC, R:CTCGAACCACATCCTTCTCT) primer used and PCR
product were analyzed in 2% agarose gel.
Expression of transcription factor Oct-4, known for expression in undifferentiated cell, was analyzed by
RT-PCR. As a result, all cell lines showed expression of high level.
Expression of Telomerase Activity
The ES cell lines were analyzed at passages between 15 and 20. ES cells were lysed with lysis buffer
from TRAPeze telomerase detection kit (Intergen, USA) and incubated at 4℃ for 30min. Lysed cell
were collected in 12,000rpm, 4℃ for 20 min and supernatant were used for telomerase activity reaction.
10㎕ of supernatant were incubated in 85℃ for 10min as a heat inactivated control. PCR performed at
30℃ for 30 min, 94℃ for 30sec, 59℃30sec in 33 cycle. They were analyzed in 12.5% non denaturing
polyacrylamide gel and post stained with SYBR green for 40 min.
The established human ES cell lines expressed high levels of telomerase activity. The high level of
telomerase activity expressed by the human ES cell lines suggests that their replicative life-span will
exceed that of somatic cell. Telomerase activity, enzyme exists in telomere and known for active in
undifferentiated cell, were high in all three cell lines.
Karyotype of Human ES Cells
Cell lines were initially karyotyped at passage between 12-15. To find out karyotype of ES cells,
chromosome analysis was performed by G-banding. Three cell lines stably retained a normal karyotype.
DNA was extracted using DNeasy mini kit (Qiagen) and ethanol precipitation. Sizes of fragment were
determined automatically using Genescan software ver 2.1(PE applied Biosystems) and compared with
the ladder by Genotyper software ver2.1.
We studied STR loci D3S1358, D18S51, D2S11, D8S1179, D7S820, D13S317, D5S818 and VNTR loci.
By performing DNA fingerprinting, we confirmed that three cell lines are different and derived from
Cryopreservation or Vitrification of Embryonic Stem cell
Each of the cell lines was successfully cryopreserved and thawed. Human ES cell lines were
cryopreserved with the vitrification method using the EM grid on passage 20. The colony formation rate
after thawing in the vitrified groups were significantly lower than those of control fresh ES cell line. The
ES cell colonies in the vitrified groups were smaller after cultivation of 2 – 4 days significantly, compared
to the control fresh ES cell group. However these effects could be overcome to nonsignificant level by the
additional culture of ES cell colonies.
Human ES cells after 4 to 5 month of culture (passage20) from about 50% confluent ten four multi dishes
(about 1000 colonies) were injected into the rear leg muscles of 4-week-old male SCID-beige mice.
Eights weeks after, the resulting teratomas were examined histologically. All three cell lines produced
teratomas after injection into severe combined immunodeficient (SCID)-beige mice. Each injected mouse
formed a teratoma including endoderm, ectoderm and mesoderm-derivatives.
Since Evans and Kaufman (1981) isolated and culture mouse embryonic stem cells in vitro, many
scientists tried to establish human embryonic stem cell., J. Thomson and colleagues (1998) reported that
ES cell lines were derived from human blastocysts. Thomson proposed that the essential characteristics of
primate ES cell should include (i) derivation from the preimplantation or periimplantation embryo, (ii)
prolonged undifferentiated proliferation, (iii) stable developmental potential to form derivatives of all
three embryonic germ layers even after prolonged culture.
Reubinoff et al (2000) also succeeded in establishment of human ES cell lines from blastocysts. They
also find out that ES cells can differentiate spontaneously and into neuron cells. These results give hope
to scientist about the clinical application of ES cells.
In this research, we used frozen 2 pronuclear stage embryos, which were donated by IVF patient and
established three human embryonic stem cell lines. Inner cell masses of two of them were isolated by
immunosurgery (SNUhES 1 & 3). The other was by whole embryo culture (SNUhES3). Mouse
embryonic fibroblast (STO) was used as a feeder layer.
Human embryonic stem cell needs mouse embryonic fibroblast to maintain the undifferentiated state in
vitro. Human embryonic stem cell went into differentiation even in the presence of LIF (Reubinoff et al.).
But reducing the risk of xenograft between mouse and human in case of clinical application, people tried
to use human embryonic fibroblast or feeder cell layer. Xu et al. reported that culturing of ES cell in
undifferentiated state in conditioned medium using mouse embryonic fibroblast and succeeded. In
conditioned media using MEF, still exist the risk of xenograft and the outer part of the ES colonies
showed high degree of differentiation. Richards et al. reported that they couldn’t maintain
undifferentiated state in conditioned medium no more than 3 passage. Therefore, more studies need to be
done for ES culture using conditioned medium.
In primate, ES cell form flat colonies certain borders. Establishes cell line SNUhES 1,2, &3 all showed
morphology of the primate. To passage the cell, colonies must be include more than 10 cells. We
disassociated colonies including more than 50 cells and they attached well when replated fresh feeder
Amit et al. reported that they isolated colonies into single cells by 96well plate and formed two clones. So,
there may be a possibility of cloning human embryonic stem cells.
All embryonic stem cells include alkaline phosphatase activity and specific surface antigens but the
expression of these antigens differ between primate and mouse. ES cells of the primate express SSEA-3
and SSEA-4, differentiated cell express SSEA-1 but mouse ES cells express vice versa. In our study, all
cell line showed alkaline phosphatase activity. And they also expressed SSEA-4 but didn't express SSEA-
1 so, these facts revealed that established ES cells are undifferentiated ES cells.
ES cells showed high level of telomerase activity and transcription factor Oct-4 also expressed in ES cells.
Normal karyotype, also the characters of the undifferentiated ES cell was examined. Established ES cell
SNUhES1, 2, & 3 all express normal karyotype.
SNUhES 1 & 3 were 46, XY and SNUhES 2 was 46, XX.
Human embryonic stem cell seems promising to clinical application for the source of cell therapy. First,
this can be used for research of factors involved in developmental process of human. Second, this can be
contributed to screening of pharmacology and other toxins. It may be very handful to development of
various medicines for human. It can be very useful source for replacement of tissues damaged by
incurable disease or accident.
Recently, many scientists trying to find out the effects of growth factors and materials involved in
developmental process. Reubinoff et al. reported that they isolated spontaneously developed into neural
progenitor cell and culture into neuron cells. Assady et al. reported that they induced human embryonic
stem cells into embryoid bodies and differentiated into pancreatic β cells. Kehat et al. produced
cardiomyocyte from embryonic stem cell.
For the clinical application of human embryonic stem cells, there are many problems to be solved like
long-term culture of embryonic stem cell in vitro for mass production, specific induction of stem cells
into certain kinds of cell and signaling pathway involved of developmental process. Furthermore, to
reduce immune rejection when these cells are transplanted into human bodies, ES cells have to be
manipulating by genetic engineering for major histo compatibility complex. To practical use of ES cells,
their must be number of researches have to be done for the further use of ES cells.
Embryonic stem cells were derived from crypreserved 2 pronuclear stage embryos that have been
fertilized in vitro — in an in vitro fertilization clinic of Seoul National University Hospital — and then
donated for research purposes with informed consent of the donors and IRB approval. Over the course
of several days, the cells of the inner cell mass proliferate and begin to crowd the culture dish. When
this occurs, they are removed gently and plated into several fresh culture dishes. Embryonic stem cells
that have proliferated in cell culture for six or more months without differentiating, are pluripotent, and
appear genetically normal, are referred to as an embryonic stem cell line. At various points during the
process of growing and subculturing the stem cells, characterization of embryonic stem cells including
Octamer (Oct)-4 expression, telomerase activity, alkaline phosphatase, stage specific embryonic antigen
(SSEA)-1,3,4 and DNA fingerprinting were done. We also determined whether the cells could be
subcultured after freezing, thawing, and replating. We confirmed whether the human embryonic stem
cells are pluripotent or not by injecting the cells into an immunosuppressed mouse to test for the
formation of a benign tumor called a teratoma. Teratomas typically contain a mixture of many
differentiated or partly differentiated cell types — an indication that the embryonic stem cells are capable
of differentiating into multiple cell types.
PART II. Cloned human Embryonic Stem Cell
The isolation of pluripotent human embryonic stem cells and breakthroughs in somatic cell nuclear
transfer (SCNT) in mammals have raised the possibility of performing human SCNT to generate
potentially unlimited sources of undifferentiated cells for use in research, with potential applications in
tissue repair and transplantation medicine.
We report the derivation of a pluripotent embryonic stem cell line (SCNT-hES-1) from a cloned human
embryonic blastocyst. Fresh oocytes were donated by healthy women for the purpose of SCNT stem cell
derivation for therapeutic cloning research and its application. A total of 242 oocytes were aspirated from
16 volunteers after ovarian stimulation. Autologus SCNT was performed; that is, the donor’s own
cumulus cell, isolated from the cumulus-oocyte complex, was transferred back into the donor’s own
enucleated oocytes. Without any report of an efficient protocol for human SCNT, several critical steps
had to be optimized, including reprogramming time, activation method, and in vitro culture conditions. A
total of 30 SCNT-derived balstcysts were cultured, 20 ICMs were isolated by immunosurgical removal of
the trophoblast, and one ES cell line (SCNT-hES-1) was derived. The resulting SCNT-hES-1 cells had a
high nucleus-to-cytoplasm ratio and prominent nucleoli. The SCNT-hES-1 cell line was mechanically
passaged by dissociation 5 to 7 days and successfully maintained its undifferentiated morphology after
continuous proliferation for more than 70 passages, while still maintaining a normal female (XX)
karyotype. Furthermore, The SCNT-hES-1 cells expressed ES cell markers such as alkaline phosphatase,
SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4, but not SSEA-1. Clumps of the cells were cultured
in vitro in suspension to form embyoid bodies. 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 were injected into the
testes of severe combined immunodeficient (SCID) mice, teratomas were obtained 6 to 7 weeks after
injection. The resulting teratomas contained tissue representative of all three germ layers. The DNA
finger printing analysis with human short tandem repeat (STR) markers indicates that the cell line
originated from the cloned blastocysts reconstructed from the donor cells, not from parthenogenetic
activation. Reverse transcription polymerase chain reaction (RT-PCR) amplification for paternally
expressed (hSNRPN and ARH1) and maternally expressed (UBE 3A and H19) genes further confirmed
that the cell line originated from the donor cells. We provide three lines of evidence supporting the
nuclear transfer origins of the SCNT-hES-1 cell line: 1) DNA extraction was verified for each of the 242
enucleated oocytes, 2) DNA fingerprinting showed heterozygous, not homozygous, chromosomes, and 3)
RT-PCR showed biparental and not unimaternal, expression of imprinted genes. In our study, one SCNT-
hES cell lines derived from 20 ICMs. It remains to be determined whether this low efficiency is due to
faulty reprogramming of the somatic cells or to subtle variations in our experimental procedures.
Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem cell
lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol.
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall
VS et al. Embryonic stem cell lines derived from human blastocysts. Science.
Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA et al.
Isolation of a primate embryonic stem cell line. PNAS USA. 1995;92:7844-7848.