Tor cocktail showed a 3.3-fold increase in the proportion of blastocyst-stage-embryos. The ability of these paracrine/ autocrine factors to promote development of early human embryos is consistent with findings showing zygote genome activation in human embryos at 4- to 8-cell stages on day 3 after fertilization when the expression of these growth factors begun to increase [26]. In the present combination treatment protocol, several distinct signaling pathways could be activated by the autocrine/paracrine factors used: EGF, IGF-I and BDNF bind to respective receptor tyrosine kinases to activate downstream phophotidyinositol-3-kinase-Akt signaling, CSF1 and GM-CSF interact with type I cytokine receptors to activate the downstream JAK/STAT pathway, whereas GDNF and artemin interact with glycosylphosphatidyl- inositol-anchored receptors to activate downstream cRET and Src kinase pathways [27]. Although the fresh tri-pronuclear zygotes used here were treated with five growth factors due to reagent availability, thawed normallyfertilized and SCNT embryos were treated with seven growth factors. It is likely that these divergent pathways exert overlapping and redundant actions on early embryo development and not all growth factors are needed for optimal embryo growth. Successful implantation of the blastocyst is essential for reproduction. Implantation of blastocysts is a well-organized process regulated by multiple growth factors and cytokines [28]. We demonstrated the facilitatory effects of key growth factors to promote blastocyst outgrowth. The trophectoderm cells of blastocysts differentiate during embryonic development to form the invasive trophoblasts that mediate implantation of embryos into the uterine wall. The 298690-60-5 outgrowth of trophoblast cells from cultured blastocysts is believed to reflect the proper differentiation of the embryo, important for trophoblast invasion of the endometrial stroma during implantation in utero [38,39]. Although blastocyst transfer is effective to select the best quality embryos with high implantation potential, overall implantation rate is ,30 [29], suggesting human embryo transfer might be improved. Due to the low amount of liquid in the uterine cavity, factors included in the transfer media could be retained in high concentrations. Indeed, embryo transfer in medium containing hyaluronan is effective in improving implantation rates in patients with recurrent implantation failure [30,31,32].Hyaluronan is the major glycosaminoglycan present in follicular, oviductal and uterine fluids and presumably promotes 
embryo ndometrial interactions during the initial phases of implantation. Because key growth factors promoted blastocyst outgrowth in vitro, future supplementation of embryo transfer media with key growth factors could also promote implantation during embryo transfer.Generating an autologous patient-specific embryonic stem cell line from SCNT embryos holds great promise for the treatment of degenerative human diseases. Successful derivation of embryonic stem cell lines following SCNT has been reported in mouse [44], rabbit [45], and Dimethylenastron non-human primates [46]. However, the efficiency for the production of 
embryonic stem cell lines following SCNT is still low (,2 ), particularly when adult somatic cells were used as the donor karyoplasts. Although many embryonic stem cell lines have been derived from surplus human blastocysts [47,48], no human cell-lines have been generated following SCNT. Among the many compo.Tor cocktail showed a 3.3-fold increase in the proportion of blastocyst-stage-embryos. The ability of these paracrine/ autocrine factors to promote development of early human embryos is consistent with findings showing zygote genome activation in human embryos at 4- to 8-cell stages on day 3 after fertilization when the expression of these growth factors begun to increase [26]. In the present combination treatment protocol, several distinct signaling pathways could be activated by the autocrine/paracrine factors used: EGF, IGF-I and BDNF bind to respective receptor tyrosine kinases to activate downstream phophotidyinositol-3-kinase-Akt signaling, CSF1 and GM-CSF interact with type I cytokine receptors to activate the downstream JAK/STAT pathway, whereas GDNF and artemin interact with glycosylphosphatidyl- inositol-anchored receptors to activate downstream cRET and Src kinase pathways [27]. Although the fresh tri-pronuclear zygotes used here were treated with five growth factors due to reagent availability, thawed normallyfertilized and SCNT embryos were treated with seven growth factors. It is likely that these divergent pathways exert overlapping and redundant actions on early embryo development and not all growth factors are needed for optimal embryo growth. Successful implantation of the blastocyst is essential for reproduction. Implantation of blastocysts is a well-organized process regulated by multiple growth factors and cytokines [28]. We demonstrated the facilitatory effects of key growth factors to promote blastocyst outgrowth. The trophectoderm cells of blastocysts differentiate during embryonic development to form the invasive trophoblasts that mediate implantation of embryos into the uterine wall. The outgrowth of trophoblast cells from cultured blastocysts is believed to reflect the proper differentiation of the embryo, important for trophoblast invasion of the endometrial stroma during implantation in utero [38,39]. Although blastocyst transfer is effective to select the best quality embryos with high implantation potential, overall implantation rate is ,30 [29], suggesting human embryo transfer might be improved. Due to the low amount of liquid in the uterine cavity, factors included in the transfer media could be retained in high concentrations. Indeed, embryo transfer in medium containing hyaluronan is effective in improving implantation rates in patients with recurrent implantation failure [30,31,32].Hyaluronan is the major glycosaminoglycan present in follicular, oviductal and uterine fluids and presumably promotes embryo ndometrial interactions during the initial phases of implantation. Because key growth factors promoted blastocyst outgrowth in vitro, future supplementation of embryo transfer media with key growth factors could also promote implantation during embryo transfer.Generating an autologous patient-specific embryonic stem cell line from SCNT embryos holds great promise for the treatment of degenerative human diseases. Successful derivation of embryonic stem cell lines following SCNT has been reported in mouse [44], rabbit [45], and non-human primates [46]. However, the efficiency for the production of embryonic stem cell lines following SCNT is still low (,2 ), particularly when adult somatic cells were used as the donor karyoplasts. Although many embryonic stem cell lines have been derived from surplus human blastocysts [47,48], no human cell-lines have been generated following SCNT. Among the many compo.
