Blastomeres of 8-cell mouse embryos differ in their ability to generate embryonic stem cells and produce lines with different transcriptional signatures.

Embryonic stem cell (ESC) derivation from single blastomeres of 8-cell mouse embryos results in lower derivation rates than that from whole blastocysts, raising a biological question about the developmental potential of sister blastomeres. We aimed to assess the ability of 8-cell blastomeres to produce epiblast cells and ESC lines after isolation, and the properties of the resulting lines. Our results revealed unequal competence among sister blastomeres to produce ESC lines. At least half of the blastomeres possess a lower potential to generate ESCs, although culture conditions and blastomeres plasticity can redirect their non-pluripotent fate towards the epiblast lineage, allowing us to generate up to seven lines from the same embryo. Lines originated from the same embryo segregated into two groups according to their transcriptional signatures. While the expression of genes related to pluripotency and development was higher in one group, no differences were found in their trilineage differentiation ability. These results may help to improve our understanding of the ESC derivation process from single blastomeres and cell fate determination in the preimplantation mouse embryos.

Efficient generation of embryonic stem cells from single blastomeres of cryopreserved mouse embryos in the presence of signalling modulators.

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.

Wnt pathway modulation generates blastomere-derived mouse embryonic stem cells with different pluripotency features.

PURPOSE: This study aimed to determine the role of Wnt pathway in mouse embryonic stem cell (mESC) derivation from single blastomeres isolated from eight-cell embryos and in the pluripotency features of the mESC established. METHODS: Wnt activator CHIR99021, Wnt inhibitor IWR-1-endo, and MEK inhibitor PD0325901 were used alone or in combination during ESC derivation and maintenance from single blastomeres biopsied from eight-cell embryos. Alkaline phosphatase activity, FGF5 levels, expression of key pluripotency genes, and chimera formation were assessed to determine the pluripotency state of the mESC lines. RESULTS: Derivation efficiencies were highest when combining pairs of inhibitors (15-24.7%) than when using single inhibitors or none (1.4-10.1%). Full naïve pluripotency was only achieved in CHIR- and 2i-treated mESC lines, whereas IWR and PD treatments or the absence of treatment resulted in co-existence of naïve-like and primed-like pluripotency features. IWR + CHIR- and IWR + PD-treated mESC displayed features of primed pluripotency, but IWR + CHIR-treated lines were able to generate germline-competent chimeric mice, resembling the predicted properties of formative pluripotency. CONCLUSION: Wnt and MAPK pathways have a key role in the successful derivation and pluripotency features of mESC from single precompaction blastomeres. Modulation of these pathways results in mESC lines with various degrees of naïve-like and primed-like pluripotency features.