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: 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 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. 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In this review, we describe the derivation and characterization of ES cells and cloned ES cells. Furthermore,