Fig. 2

The role of signaling pathways in self-renewal and differentiation of in vitro pluripotent stem cell populations. Embryonic stem cells (ESCs) represent a naïve state of pluripotency similar to the pre-implantation epiblast (Epi). ESCs are routinely maintained in a self-renewing state in serum (a source of BMP) and LIF (SL). Under these conditions, ESC cultures are heterogeneous and contain subpopulations of lineage-primed cells (yellow and green cells) i.e. cells that coexpress germ layer markers alongside pluripotency markers, and are biased in differentiation towards particular lineages. A more homogeneous ESC state can be generated by blocking FGF signaling using a MEK inhibitor (PD0325901), and activating Wnt signaling using a GSK3 inhibitor (CHIR99021), a condition known as ‘2i’. In these conditions, self-renewal occurs in the absence of external signals, although cell propagation is enhanced in the presence of Wnt pathway activity through CHIR99021. This state is referred to as the naïve or “ground state” of pluripotency. ESCs can be pushed further along the differentiation trajectory by culturing in the presence of FGF and Activin (FA) for 48 hours to generate a cell state referred to as Epi-like cells (EpiLCs). This is a transient cell state, and it is unknown whether self-renewing EpiLCs can be captured by the addition of other factors. It is also not known whether EpiLCs are a homogeneous population of cells. Upon further differentiation in FA over multiple passages, cells resemble a primed state of pluripotency akin to the later post-implantation Epi, referred to as epiblast stem cells (EpiSCs). While FA promotes differentiation of ESCs and EpiLCs, it promotes EpiSC self-renewal. EpiSCs can be derived from ESCs in culture or directly from embryos with FA. When grown in FA, EpiSCs, like ESCs in SL, are heterogeneous and contain lineage-primed populations. While activation of Wnt signaling promotes a naïve ground state of self-renewal, inhibition of Wnt signaling promotes a more homogeneous primed ground state of self-renewal. Therefore cells in naïve and primed pluripotent states respond to signaling factors with opposite outcomes, Wnt and BMP promote self-renewal of the naïve state but differentiation of the primed state of pluripotency and conversely FA promote differentiation of the naïve state but self-renewal of the primed state of pluripotency. Addition of BMP4 and WNT3A in combination with FA stimulates further differentiation of EpiSCs into PS-derived mesoderm and endoderm, while in the absence of FA, BMP or Wnt EpiSCs differentiate to neurectoderm [127]. EpiLCs are the only pluripotent state that has been shown to efficiently generate primordial germ cell-like cells (PGCLCs). Presumably, ESCs have not yet acquired this capacity, while EpiSCs have lost it. Cells within the dashed box are within the pluripotency spectrum while cells outside have differentiated. Blue arrows indicate self-renewal. Orange arrows denote the direction of differentiation along the developmental trajectory