Detailed method description for non-invasive monitoring of differentiation sta- tus of human embryonic stem cells

Abstract The (non-)differentiation status of human embryonic stem cells (hESC) is usually analyzed by determination of key pluripotency defining markers (OCT4, Nanog, SOX2, …) by means of RT-qPCR, flow cytometry (FC) and immunostaining. Despite proven usefulness of these techniques, their destructive nature makes it impossible to follow-up on the same hESC colonies during several days, leading to a loss of information. In 2003, an OCT4-eGFP Knock-In hESC line to monitor OCT4-expression was developed and commercialized. However, to the best of our knowledge, the use of fluorescence microscopy (FM) for monitoring the OCT4-eGFP expression of these cells without sacrificing them has not been described to date. Here, we describe such a method in detail, emphasizing both its resolving power and complementary nature to FC, as well as the potential pitfalls in standardizing the output of the FM measurements. The potential of the method is demonstrated by comparison of hESC cultured in several conditions, both feeder-free (vitronectin) and grown on feeder cells (mouse embryonic fibroblasts (MEF)). Keywords: OCT4-eGFP Knock-In hESC; fluorescence microscope; flow cytometry; OCT4; MEF CM