ABSTRACT
Floating spheroidal aggregates (aggregomes) of mouse embryonic stem cells (mESCs) can develop into polarized/elongated organoids, namely gastruloids. Here we report a high-performing assay to measure gastruloids formation efficiency (GFE), i.e. the fraction of gastruloid-developing aggregomes. By exploiting this procedure, we provide morphological and molecular evidence that gastruloid development relies on Cripto. We also demonstrate that GFE decreases as pluripotency progresses from naïve to primed state. Indeed, naïve ESC-derived aggregomes efficiently elongate (GFE≥95%), while primed EpiSCs fail to aggregate and consequently to generate gastruloids (GFE=0%). Conversely, while early-primed EpiLCs properly aggregate, EpiLC-derived aggregomes are mostly abortive (GFE=0%). Unlike EpiLCs, L-Proline-treated ESCs (PiCs) generate productive aggregomes (GFE≥50%), which however begin to elongate earlier and generate smaller gastruloids that appear more differentiated. Like EpiLCs, PiCs are competent to differentiate into primordial germ cell-like cells (PGCLCs), suggesting that PiCs capture an EpiLC-like state with unique competence for both gastruloid formation and differentiation into PGCLCs. Thus we propose GFE assay as a simple and robust in vitro method to discriminate different phenotypic/functional states of the pluripotency continuum.
