DUSP6 is a memory retention feedback regulator of ERK signaling for cellular resilience of human pluripotent stem cells in response to dissociation

Abstract

Cultured human pluripotent stem cells (hPSCs) grow as colonies that require breakdown into small clumps for further propagation. Although cell death mechanism by single-cell dissociation of hPSCs has been well defined, how hPSCs respond to the deadly stimulus and recover the original status remains unclear. Here we show that dissociation of hPSCs immediately activates ERK, which subsequently activates RSK and induces DUSP6, an ERK-specific phosphatase. Although the activation is transient, DUSP6 expression persists days after passaging. DUSP6 depletion using the CRISPR/Cas9 system reveals that DUSP6 suppresses the ERK activity over the long term. Elevated ERK activity by DUSP6 depletion increases both viability of hPSCs after single-cell dissociation and differentiation propensity towards mesoderm and endoderm lineages. These findings provide new insights into how hPSCs respond to dissociation in order to maintain pluripotency.

Polydeoxyribonucleotide exerts opposing effects on ERK activity in human skin keratinocytes and fibroblasts

“…To investigate the molecular mechanism involved in PDRN induction, HDFs (2×10 5 cells/well) and HEKs (4×10 5 cells/well) were seeded in six-well culture plates in growth medium for 24 h. Cells were serum-starved for 16 h before supplementation with PDRN for 0, 5, 15, 30, 60 and 120 min. Confirming changes in MAPK phosphorylation kinetics under stimulation within 2 h has been widely used as a method to monitor MAPK activation in vitro ( 23 , 24 ). Protein expression levels of phosphorylated ERK1/2, JNK and p38 were determined by immunoblotting.…”