A high-throughput multiplexed screening assay for optimizing serum-free differentiation protocols of XXXX embryonic stem cells.

@entity1 @entity13062 @entity7 @entity540 @entity436
Serum-free differentiation protocols of @entity1 embryonic stem cells (hESCs) offer the ability to maximize reproducibility and to develop clinically applicable therapies. We developed a high-throughput, 96-well plate, four-color flow cytometry-based assay to optimize differentiation media cocktails and to screen a variety of conditions. We were able to differentiate hESCs to all three primary germ layers, screen for the effect of a range of activin A, @entity13062 , and @entity7 concentrations on endoderm and mesoderm differentiation, and perform RNA-interference (RNAi)-mediated knockdown of a reporter gene during differentiation. Cells were seeded in suspension culture and embryoid bodies were induced to differentiate to the three primary germ layers for 6 days. Endoderm ( @entity436 (+)KDR(-)), mesoderm (KDR(+)SSEA-3(-)), and ectoderm (SSEA-3(+)NCAM(+)) differentiation yields for H9 cells were 80    11, 78    7, and 41    9%, respectively. Germ layer identities were confirmed by quantitative PCR. Activin A, @entity13062 , and @entity540 drove differentiation, with increasing concentrations of activin A inducing higher endoderm yields and increasing @entity13062 inducing higher mesoderm yields. @entity7 drove lateral mesoderm differentiation. RNAi-mediated knockdown of constitutively expressed red fluorescent protein did not affect endoderm differentiation. This assay facilitates the development of serum-free protocols for hESC differentiation to target lineages and creates a platform for screening small molecules or RNAi during ESC differentiation.
