Simple differentiation method using FBS identifies DUSP6 as a marker for fine-tuning of FGF-ERK signaling activity in human pluripotent stem cells

Assessment of differentiation potential is a basic requirement to obtain qualified human pluripotent stem cells (hPSCs). Here, we report a simple differentiation method using fetal bovine serum (FBS) to estimate differentiation potential and propensity of hPSCs. PluriTest using RNA-sequencing showed that cells differentiated after treatment with 5% FBS. Expression patterns of three germ layer markers revealed that cells cultured in Knockout Serum Replacement-containing medium (KSR) with mouse feeder cells had higher differentiation potential than cells cultured in a chemically defined medium (E8) with recombinant matrix proteins, especially into the mesoderm and endoderm lineages. Analysis of differentially expressed genes between KSR and E8 identified DUSP6 as a marker for where cells had been cultured. Expression of DUSP6 correlated with FGF-ERK signaling activity. Fine-tuning of FGF-ERK signaling activity to a range that can shut down DUSP6 transcription but sustain NANOG transcription partially increased the differentiation potential. Our data suggest that differentiation with 5% FBS is good to estimate differentiation potential and propensity at the early stage, and that DUSP6 is an excellent marker to monitor ERK signaling activity. Overall design: Two biological replicates were analyzed for each point of differentiation of human pluripotent stem cells cultured in two widely used media. For differentiation with 5% FBS, hFSiPS1 cells were cultured KSR and E8 media for 3 weeks and differentiated with 5% FBS. Cells were harvested after 1, 2, 3 weeks. For culture media, hPSCs, hFSiPS1, hFmiPS2, SNUhES31, H9, were cultured for 13 weeks and harvested at 3 weeks and 13 weeks.