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

被引:208
作者
Burridge, Paul W.
Anderson, David
Priddle, Helen
Munoz, Maria D. Barbadillo
Chamberlain, Sarah
Allegrucci, Cinzia
Young, Lorraine E.
Denning, Chris [1 ]
机构
[1] Univ Nottingham, Queens Med Ctr, Genet Inst, Nottingham NG7 2UH, England
[2] Univ Nottingham, Queens Med Ctr, Wolfson Ctr Stem Cells Tissue Engn & Modelling, Sch Human Dev, Nottingham NG7 2UH, England
[3] Univ Nottingham, Queens Med Ctr, Res & Treatment Unit, Nottingham NG7 2UH, England
基金
英国生物技术与生命科学研究理事会; 英国医学研究理事会;
关键词
human embryonic stem cells; embryoid body; forced aggregation; differentiation; cardiomyocytes; activin A; basic fibroblast growth factor;
D O I
10.1634/stemcells.2006-0598
中图分类号
Q813 [细胞工程];
学科分类号
摘要
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 (alpha 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-as a line that can respond to cardiomyogenic stimulation.
引用
收藏
页码:929 / 938
页数:10
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