Veterinary Science Human embryonic stem cells and therapeutic cloning Woo Suk Hwang1,2,3,*, Byeong Chun Lee1,2, Chang Kyu Lee3, Sung Keun Kang1,2 1Department of Theriogenology and Biotec
Trang 1Veterinary Science
Human embryonic stem cells and therapeutic cloning
Woo Suk Hwang1,2,3,*, Byeong Chun Lee1,2, Chang Kyu Lee3, Sung Keun Kang1,2
1Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
2The Xenotransplantation Research Center, Seoul National University Hospital, Seoul 110-744, Korea
3School 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
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: hwangws@snu.ac.kr
Review
Trang 2and 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
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
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,
[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
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
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
Trang 3culture 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
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
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
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
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
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
[33] showed similar results using mouse cumulus cells as nuclear donors and demonstrated that these mouse nuclear
that dedifferentiated cloned mouse ES cells derived from nuclear transfer of cumulus cells can go to the germline and
Trang 4produce offspring [79] The same group also demonstrated
that neurons derived from somatic-cell-cloned-ES cells can
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
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
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
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-cell Compacted
morula Blastocyst
1st set
10 µM Ionophore 6-DMAP 2 G 1.2 hmSOFaa 16 16 (100) 4 (25) 4 (25)
2nd set
10 µM Ionophore 6-DMAP 2 G 1.2 hmSOFaa 16 16 (100) 5 (31) 3 (19)
3rd set
10 µM Ionophore 6-DMAP 2 G 1.2 hmSOFaa 16 16 (100) 4 (25) 3 (19)
4th set 10 µM Ionophore 6-DMAP 2 G 1.2 hmSOFaa 66 62 (93) 24 (36) 19 (29)
followed by 2 mM 6-dimethylaminopurine (DMAP) treatment for 4 hrs.
Trang 5albumin 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%
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 contrast 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 and C)
Trang 6SSEA-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
vitro and in vivo For embryoid body formation, clumps of
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
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
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
Trang 7successful 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
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
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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