A transient DMSO treatment increases the differentiation potential of human pluripotent stem cells through the Rb family

The propensity for differentiation varies substantially across human pluripotent stem cell (hPSC) lines, greatly restricting the use of hPSCs for cell replacement therapy or disease modeling. Here, we investigate the underlying mechanisms and demonstrate that activation of the retinoblastoma (Rb) pathway in a transient manner is important for differentiation. In prior work, we demonstrated that pre-treating hPSCs with dimethylsulfoxide (DMSO) before directed differentiation enhanced differentiation potential across all three germ layers. Here, we show that exposure to DMSO improves the efficiency of hPSC differentiation through Rb and by repressing downstream E2F-target genes. While transient inactivation of the Rb family members (including Rb, p107, and p130) suppresses DMSO’s capacity to enhance differentiation across all germ layers, transient expression of a constitutively active (non-phosphorylatable) form of Rb increases the differentiation efficiency similar to DMSO. Inhibition of downstream targets of Rb, such as E2F signaling, also promotes differentiation of hPSCs. More generally, we demonstrate that the duration of Rb activation plays an important role in regulating differentiation capacity.