title: Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability.
abstract: Although all human ESC (hESC) lines have similar morphology, express key pluripotency markers, and can differentiate toward primitive germ layers in vitro, the lineage-specific developmental potential may vary between individual lines. In the current study, four hESC lines were cultured in the same feeder-free conditions to provide a standardized platform for interline analysis. A high-throughput, forced-aggregation system involving centrifugation of defined numbers of hESCs in V-96 plates (V-96FA) was developed to examine formation, growth, and subsequent cardiomyocyte differentiation from >22,000 EBs. Homogeneity of EBs formed by V-96FA in mouse embryo fibroblast-conditioned medium was significantly improved compared with formation in mass culture (p < .02; Levene's test). V-96FA EB formation was successful in all four lines, although significant differences in EB growth were observed during the first 6 days of differentiation (p = .044 to .001; one-way analysis of variance [ANOVA]). Cardiomyocyte differentiation potential also varied; 9.5% +/- 0.9%, 6.6% +/- 2.4%, 5.2% +/- 3.1%, and 1.6% +/- 1.0% beating EBs were identified for HUES-7, NOTT2, NOTT1, and BG01, respectively (p = .008; one-way ANOVA). Formation of HUES-7 V-96FA EBs in defined medium containing activin A and basic fibroblast growth factor resulted in 23.6% +/- 3.6% beating EBs, representing a 13.1-fold increase relative to mass culture (1.8% +/- 0.7%), consistent with an observed 14.8-fold increase in MYH6 (alphaMHC) expression by real-time polymerase chain reaction. In contrast, no beating areas were derived from NOTT1-EBs and BG01-EBs formed in defined medium. Thus, the V-96FA system highlighted interline variability in EB growth and cardiomyocyte differentiation but, under the test conditions described, identified HUES-7 as a line that can respond to cardiomyogenic stimulation.
authors.affiliation: Wolfson Centre for Stem Cells, Tissue Engineering and Modelling, School of Human Development, University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Burridge
authors.forename: David
authors.initials: D
authors.lastname: Anderson
authors.forename: Helen
authors.initials: H
authors.lastname: Priddle
authors.forename: Maria D
authors.initials: MD
authors.lastname: Barbadillo Muñoz
authors.forename: Sarah
authors.initials: S
authors.lastname: Chamberlain
authors.forename: Cinzia
authors.initials: C
authors.lastname: Allegrucci
authors.forename: Lorraine E
authors.initials: LE
authors.lastname: Young
authors.forename: Chris
authors.initials: C
authors.lastname: Denning
mesh_terms: D000818:Animals; D002449:Cell Aggregation; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002460:Cell Line; D003470:Culture Media; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006801:Humans; D007621:Karyotyping; D008285:Major Histocompatibility Complex; D051379:Mice; D032342:Muscle Cells / Q000166:cytology*; D009206:Myocardium / Q000166:cytology*
chemical_list: D003470:Culture Media