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 lineagespecific 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 highthroughput, 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 (␣MHC) 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. STEM CELLS 2007;25:929 -938 Disclosure of potential conflicts of interest is found at the end of this article.

Hydrophobic surfaces for enhanced differentiation of embryonic stem cell-derived embryoid bodies

“…This even distribution, combined with a reduction in debris and larger aggregates, translated to morphologically uniform EBs after 4 days of culture (Fig. S2 C-H), suggesting that PDMS surfaces may better support EB development (22)(23)(24).…”

Accurate nanoelectrode recording of human pluripotent stem cell-derived cardiomyocytes for assaying drugs and modeling disease

“…First, hPSC-CMs are intrinsically heterogeneous, consisting of three subpopulations: atrial-, ventricular-, and nodal-like 10 . Furthermore, electrophysiology measurements have shown that the shapes of action potentials vary significantly between studies and within studies among different cell lines and differentiation methods 11,12 . Therefore, the high heterogeneity among hPSC-CMs requires that a large number of cells be analyzed to generate statistically meaningful conclusions.…”

Controlled embryoid body formation via surface modification and avidin–biotin cross-linking

“…Researchers employ a myriad of aggregation methods for EB formation which promote natural aggregation or artificially force cell-cell interactions, such as rotary mass suspension (Abilez et al 2006;Niebruegge et al 2008) or microwell centrifugation (Burridge et al 2007;Moeller et al 2008), respectively. Many differences exist between methods regarding aggregation efficiency (Carpenedo et al 2007).…”