title: A high-throughput multiplexed screening assay for optimizing serum-free differentiation protocols of human embryonic stem cells.
abstract: Serum-free differentiation protocols of human 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, BMP4, and VEGF 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 (CXCR4(+)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, BMP4, and bFGF drove differentiation, with increasing concentrations of activin A inducing higher endoderm yields and increasing BMP4 inducing higher mesoderm yields. VEGF 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.
authors.affiliation: Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Joel T
authors.initials: JT
authors.lastname: Outten
authors.forename: Xin
authors.initials: X
authors.lastname: Cheng
authors.forename: Paul
authors.initials: P
authors.lastname: Gadue
authors.forename: Deborah L
authors.initials: DL
authors.lastname: French
authors.forename: Scott L
authors.initials: SL
authors.lastname: Diamond
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D058732:Embryoid Bodies / Q000166:cytology / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods; D005855:Germ Layers / Q000166:cytology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D051379:Mice; D034622:RNA Interference
chemical_list: D015415:Biomarkers; D016895:Culture Media, Serum-Free