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

¹ Wolfson Centre for Stem Cells, Tissue Engineering and Modelling, School of Human Development, University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom; Institute of Genetics University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom
² Institute of Genetics University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom
³ Wolfson Centre for Stem Cells, Tissue Engineering and Modelling, School of Human Development, University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom; NURTURE, University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom
⁴ NURTURE, University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom

^* To whom correspondence should be addressed. E-mail: chris.denning{at}nottingham.ac.uk.

	Abstract

While all human embryonic stem cell (hESC) lines have similar morphology, express key pluripotency markers and can differentiate towards 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 inter-line 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 embryoid bodies (EBs). Homogeneity of EBs formed by V-96FA in mouse embryo fibroblast conditioned medium (CM) was significantly improved compared to formation in mass culture (P<0.02; 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=0.044 to 0.001; one-way 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 (P=0.008; one-way Anova). Formation of HUES-7 V-96FA EBs in defined medium containing activin A and bFGF 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 (MHC) expression by real-time PCR. In contrast, no beating areas were derived from NOTT1-EBs and BG01-EBs formed in defined medium. Thus, the V-96FA system highlighted inter-line 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.

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