Enhanced generation of human embryonic stem cells from single blastomeres of fair and poor-quality cleavage embryos via inhibition of glycogen synthase kinase and Rho-associated kinase signaling

Could selected pluripotency-enhancing small molecules (SMs) lead to efficient derivation of human embryonic stem cells (hESCs) from cleavage embryos-derived single blastomeres (SBs)? Inhibition of glycogen synthase kinase (GSK3) and Rho-associated kinase (ROCK) signaling can enhance the derivation of hESCs from cleavage embryo-derived SBs. Parameters involved in sustaining the pluripotency of biopsied blastomeres for generating hESCs without causing injury to a viable embryo have remained obscure. This research seeks to improve the culture conditions for increasing the efficiency of deriving hESCs from SBs from cleavage-stage embryos by using SMs. In order to identify SMs which may enhance hESC generation from SBs, 11 pluripotency-enhancing SMs were screened and CHIR99021 (CH), a GSK3 inhibitor, was selected. To optimize culture condition in hESC generation from SMs, we used ROCK inhibitor Y27632 (Y) and basic fibroblast growth factor in combination with CH or its alternative, Kenpaullone, in different time courses over 12 days. We also assessed a critical time point for CH Y treatment of cleavage embryos from 4- to 8-cell embryo. In total, 224 embryos and 1607 SBs were used in the study. Blastomeres of fair and poor-quality from 6- to 8-cell stage human embryos were mechanically dispersed and individually seeded into a 96-well plate that was precoated with mitotically inactivated feeder cells. Derivation of hESC line from each SB was carried out in hESC defined medium supplemented with SMs. Randomly selected hESC lines were evaluated by immunostaining for pluripotency markers, karyotype analysis and differentiation potential into the three embryonic germ layer derivatives. We found that 3 M CH was the only SM that was capable of directing SBs from fair and poor-quality 68-cell embryos into hESC lines. The application of hESC-conditioned medium had no additive effect on hESC establishment from SBs. Also, we indicated that CH combined with Y improved hESC generation efficiency by up to 31. By using of Kenpaullone as an alternative to CH, we confirmed the involvement of GSK3 inhibition in hESC derivation from SBs. Interestingly, by treatment of 4-cell embryos, these SMs could enhance the derivation efficiency of SB-derived hESC lines up to 73 and the maximum number of hESC lines from SBs of one embryo was achieved in this state. The low quality of the embryos used in this study most likely had an effect on hESC generation. Furthermore, although we attempted to minimize any differences in inter-embryo quality, we cannot exclude the possibility that small differences in starting quality between embryos may have contributed to the differences observed, other than the addition of SMs. This approach would allow the establishment of autogeneic or allogeneic matched cells from embryos fertilized in vitro without destroying them. This study was financially supported by the National Elite Foundation and the Royan Institute for Stem Cell Biology and Technology. The authors have no conflict of interest to declare.