Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability

Paul W. Burridge, David Anderson, Helen Priddle, Maria D. Barbadillo Muñoz, Sarah Chamberlain, Cinzia Allegrucci, Lorraine E. Young, Chris Denning*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

218 Scopus citations

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 (αMHC) 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.

Original languageEnglish (US)
Pages (from-to)929-938
Number of pages10
JournalStem Cells
Volume25
Issue number4
DOIs
StatePublished - 2007

Keywords

  • Activin A
  • Basic fibroblast growth factor
  • Cardiomyocytes
  • Differentiation
  • Embryoid body
  • Forced aggregation
  • Human embryonic stem cells

ASJC Scopus subject areas

  • Molecular Medicine
  • Developmental Biology
  • Cell Biology

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