A Study on the Induction of Mesoderm Differentiation of Mouse Embryonic Stem Cell in Serum Free Culture.

(Introduction) Several products which can maintain the growth of ES (Embryonic stem) cell with no feeder cells or fetal calf serum (FCS) is now commercially available. Culti Cell ^®(CC) can be a substitute for FCS and has ability of mainitaining mouse ES cells without feeder cells. Previously, it was reported that ES cell differentiate into FLk (fetal liver kinase) positive cells derived from mesoderm in methylcellulose and FCS. We investigated whether ES cell is able to differentiate into Flk positive cell in CC and methylcellulose without FCS.

(Materials and Methods) Thawed ES cells were cultured with methylcellulose and CC in the absence of FCS and feeder cells. EB (Embryoid body) formation was completed after 7 days. The separated cell from EB was adjusted to 1×10⁴/ml. These ES cells were cultured with 15% CC and 0.8% methylcellulose for 7 days at 37°C in 5% CO₂. The expression of Flk and mesoderm differentiation was analyzed with flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) at day 1, 2, 3, 5 and 7. Oligonucleotide primers for Flk1 and brachyury were used for RT-PCR. In addition, the expression of CD31 was also examined with flow cytometry.

(Results) No Flk positive cells were detected in these culture periods. The expression of brachyury, which is a marker of mesoderm differentiation, was consistently observed throughout culture days. The expression of CD31 gradually diminished as the culture period increased.

(Conclusion) These results suggest that ES cell is maintained and differentiate into mesoderm in suspension culture with CC and methylcellulose, but diferentiation to Flk positive cell can not be induced in this culture condition. Further examination is necessary to clarify unidentified factors for differentiation to Flk positive cell in serum free culture.