Expression profiling by high throughput sequencing
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.
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.