How Do I Avoid Spontaneous Differentiation of my PSC-Culture?

Ask the expert - Answer by Dr. Nadine Mellies, Application Specialist Cell Handling at Eppendorf

Pluripotent stem cells (PSCs) are per se prone to differentiate spontaneously in culture. Controlling the balance between their expansion and differentiation can be challenging, but reliable maintenance in the undifferentiated state is critical for stem cell propagation. When maintained properly, spontaneous differentiation should be <5%. PSCs can be distinguished easily from differentiated cells, which show less-defined edges, loose morphology, dark areas, and/or exhibit fibroblast- or endothelial-like morphology. Those differentiated areas must be removed from the culture. Using fully chemically defined media with appropriate hormones and growth factors can reduce the risk of serum-induced spontaneous differentiation of PSCs. Spontaneous differentiation can be further minimized with appropriate split ratios to balance the culture confluency, while cell overgrowth will trigger spontaneous differentiation, and result in loss of pluripotency and differentiation potential. Regular monitoring of PSC morphology and careful handling are therefore mandatory to maintain high-quality PSC cultures.