Josep Santaló

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.

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.

La mejora genética humana en los tiempos del CRISPR/Cas9

To assess the role of the genetic background, the culture medium supplements, and the presence of modulators of signaling pathways on mouse embryonic stem cell derivation from single blastomeres from 8-cell embryos.

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

Oocyte vitrification causes a temporary disassembly of the metaphase plate and spindle, which needs time to recover after warming. As a consequence, early post-fertilization events – such as timing of second polar body extrusion – might be altered, with unknown effects on preimplantation development, timing to pronuclear breakdown, and timing of cleavages. The aim of this study was to evaluate if differences exist among these events when comparing embryos obtained from fresh-donated versus vitrified/warmed oocytes from young women. We performed a prospective study with 201 embryos from 100 fresh and 101 vitrified/warmed oocytes that were subsequently fertilized by intracytoplasmic sperm injection. Kaplan-Meier curves of each time period were generated, in which we observed that median developmental times did not differ between embryos from fresh versus vitrified/warmed oocytes among all the metrics assessed. Thus, for young women without fertility problems, no differences exist between the timing of early developmental milestones in embryos derived from fresh or vitrified oocytes, and vitrification does not affect the preimplantation development of the resulting embryos. This article is protected by copyright. All rights reserved

Time-lapse monitoring of somatic cell nuclear transfer (SCNT) embryos may help to predict developmental success and increase birth and embryonic stem cells (ESC) derivation rates. Here, the development of ICSI fertilized embryos and of SCNT embryos, non-treated or treated with either psammaplin A (PsA) or vitamin C (VitC), was monitored, and the ESC derivation rates from the resulting blastocysts were determined. Blastocyst rates were similar among PsA-treated and VitC-treated SCNT embryos and ICSI embryos, but lower for non-treated SCNT embryos. ESC derivation rates were higher in treated SCNT embryos than in non-treated or ICSI embryos. Time-lapse microscopy analysis showed that non-treated SCNT embryos had a delayed development from the second division until compaction, lower number of blastomeres at compaction and longer compaction and cavitation durations compared with ICSI ones. Treatment of SCNT embryos with PsA further increased this delay whereas treatment with VitC slightly reduced it, suggesting that both treatments act through different mechanisms, not necessarily related to their epigenetic effects. Despite these differences, the time of completion of the third division, alone or combined with the duration of compaction and/or the presence of fragmentation, had a strong predictive value for blastocyst formation in all groups. In contrast, we failed to predict ESC derivation success from embryo morphokinetics. Time-lapse technology allows the selection of SCNT embryos with higher developmental potential and could help to increase cloning outcomes. Nonetheless, further studies are needed to find reliable markers for full-term development and ESC derivation success.