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 journalArticle

214 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

Fingerprint Dive into the research topics of 'Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability'. Together they form a unique fingerprint.

  • Cite this