Figure 2.

Experimental approaches to investigate potential totipotency. In (a,b), typical chimera assays are shown. In (a), incorporation into 8-cell stage pre-implantation mouse embryos is done by aggregation, typically referred to as ‘morula aggregation’. In this assay, contribution to lineage is based on 3D position, but should be complemented with co-immunostaining to establish at least partial molecular identity. EGFP, enhanced green fluorescent protein; FACS, fluorescence activated cell sorting; H2B, histone H2B; NLS, nuclear localization signal. In (b), incorporation is achieved through microinjection of cells into the blastocoel of early blastocysts, followed by implantation and analysis of the conceptus, typically at embryonic day (E) 9.5. In this assay, contribution is based on expression of a fluorescent reporter in the placenta. These are difficult experiments, often hindered by the fact that the placenta is highly autofluorescent, and often it is not straightforward to distinguish between placenta and the yolk sac, which is an ICM derivative. These analyses should be accompanied by a stringent analysis through sections and molecular analysis of markers from the trophoblastic derivatives of the placenta. scRNAseq, single cell RNA sequencing. In (c), cell culture strategies are shown. In (c), as suggested by Baker and Pera [53], the ability of a single cell to give rise to stem cells from the three lineages of the mature blastocyst is depicted. XEN cells, primitive endoderm-derived stem cells; TS cells, trophoblast stem cells, derived from the trophectoderm. Molecular analysis of each of these cell types for the known relevant markers should be performed. (d) A potential design to promote self-aggregation of 2CLC, and derivation of cyst-containing structures also referred to as blastocyst-like. As in (c), a molecular analysis and exploration of lineage markers should be performed. In (e,f), nuclear transfer (NT) strategies are shown. (e) The rationale behind using somatic cell nuclear transfer as an assay to test cellular plasticity, based on the observations that nuclei derived from early embryos show a highest success in generating embryos and pups upon cloning, as opposed to pluripotent stem cells. Accordingly, 2CLC nuclei show a higher success in producing clone embryos upon NT [31]. SCNT, somatic cell nuclear transfer. In (f), the schematic of an NT experiment, including the potential outcomes and implications, is shown.