Stem cell-based embryo models have allowed greater insight into peri-implantation mammalian developmental events that are otherwise difficult to manipulate due to the inaccessibility of the early embryo. The rapid development of this field has resulted in the precise roles of frequently used supplements such as N2, B27 and Chiron in driving stem cell lineage commitment not being clearly defined. Here, we investigate the effects of these supplements on embryoid bodies to better understand their roles in stem cell differentiation. We show that Wnt signalling has a posteriorising effect on stem cell aggregates and directs differentiation towards the mesoderm, as confirmed through the upregulation of posterior and mesodermal markers. N2 and B27 can mitigate these effects and up-regulate the expression of anterior markers. To control the Wnt gradient and the subsequent anterior vs. posterior fate, we make use of a BMP4 signalling centre and show that aggregates in these conditions express cephalic markers. These findings indicate that there is an intricate balance between various culture supplements and their ability to set up the anterior/posterior axis in stem cell embryo models.

Summary Statement

The complex reagents used in ’stembryo’ protocols have unclear roles in stem cell differentiation in vitro requiring further investigation. This study examines their effects on embryoid bodies.