Context Derivation of embryonic stem cells (ESC) from single blastomeres is an interesting alternative to the use of whole blastocysts, but derivation rates are lower and the requirements for successful ESC obtention are still poorly defined. Aims To investigate the effects of embryo cryopreservation and of signalling modulators present during embryo culture and/or ESC establishment on ESC derivation efficiency from single 8-cell mouse blastomeres. Method Fresh and cryopreserved 2-cell embryos were cultured and biopsied at the 8-cell stage. Single blastomeres were cultured in the presence of 2i or R2i cocktails, with or without adrenocorticotropic hormone (ACTH). We analysed ESC derivation efficiencies and characterised pluripotency genes expression and karyotype integrity of the resulting lines. We also evaluated the impact of embryo preculture with R2i on epiblast cell numbers and derivation rates. Key results The ESC generation was not compromised by embryo cryopreservation and ACTH was dispensable under most of the conditions tested. While 2i and R2i were similarly effective for ESC derivation, R2i provided higher karyotype integrity. Embryo preculture with R2i yielded increased numbers of epiblast cells but did not lead to increased ESC generation. Conclusions Our findings help to define a simplified and efficient procedure for the establishment of mouse ESC from single 8-cell blastomeres. Implications This study will contribute to improving the potential of this experimental procedure, providing a tool to investigate the developmental potential of blastomeres isolated from different embryonic stages and to reduce the number of embryos needed for ESC derivation.

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