Title
Early gene expression changes during embryonic stem cell differentiation into cardiomyocytes and their modulation by monobutyl phthalate
Author
van Dartel, D.A.M.
Pennings, J.L.A.
Hendriksen, P.J.M.
van Schooten, F.J.
Piersma, A.H.
TNO Kwaliteit van Leven
Publication year
2009
Abstract
The Embryonic Stem cell Test (EST) is an in vitro alternative test designed for the prediction of embryotoxicity. The endpoint of the test is the interference with mesoderm-derived cardiac muscle differentiation observed under the microscope as beating muscle foci. The relative subjectivity of this endpoint, as well as the applicability domain and related predictivity need further to be defined to facilitate implementation of the EST into regulatory strategies. The use of transcriptomics techniques to monitor differentiation-related gene expression changes in the EST might improve the EST in each of these aspects. Therefore, we studied the gene expression profile in embryonic stem cells (ESC) in the early phase of differentiation and its modulation by exposure to the well known embryotoxicant monobutyl phthalate (MBP). Cells were exposed from the early embryoid body stage onwards and RNA was collected after 6, 12 and 24 h of exposure. Samples were hybridized to spotted microarrays, containing 21,997-mer oligonucleotides. Differential gene expression patterns were analyzed. A total number of 43 genes that were found to be upregulated in this study as a consequence of induction of cardiomyocyte differentiation were combined in a gene set, named 'VAN_DARTEL_HEARTDIFF_24H'. Gene Set Enrichment Analysis (GSEA) comparative analysis using multiple gene set collections clearly showed that temporal changes in gene expression were functionally related to cardiomyocyte differentiation. Furthermore, exposure of embryoid bodies (EB) to MBP increased expression of pluripotency-, proliferation- and nonmesodermal differentiation-related gene sets, which indicates inhibition of mesodermal differentiation. The inhibition of mesoderm-derived cardiomyocyte differentiation by MBP exposure was most obvious through the downregulation of our novel gene set identified in this study, 'VAN_DARTEL_HEARTDIFF_24H', which specifically describes the niche of early cardiomyocyte differentiation. The gene set defined in this study might serve as a starting point for defining a dedicated gene set for early detection of embryotoxicity in the EST. Such a gene set may serve as an improved endpoint in the EST as compared to morphology, and will allow a more detailed definition of the applicability domain and predictivity of EST. © 2008 Elsevier Inc. All rights reserved. Chemicals / CAS: RNA, 63231-63-0; phthalic acid, 88-99-3; Phthalic Acids; monobutyl phthalate, 131-70-4
Subject
Alternative test method
Differentiation
Embryonic stem cells
Monobutyl phthalate
Transcriptomics
monobutyl phthalic acid
oligonucleotide
phthalic acid
RNA
Unclassified drug
Animal cell
Cell differentiation
Cell proliferation
Controlled study
Embryonic stem cell
Gene control
Heart muscle cell
Mesoderm
Microarray analysis
Modulation
Mouse
Nonhuman
Pluripotent stem cell
Animals
Cell Differentiation
Cell Proliferation
Cells, Cultured
Dose-Response Relationship, Drug
Embryonic Stem Cells
Gene Expression Profiling
Gene Expression Regulation, Developmental
Gene Regulatory Networks
Mice
Muscle Contraction
Myocytes, Cardiac
Oligonucleotide Array Sequence Analysis
Phthalic Acids
Risk Assessment
Time Factors
Toxicity Tests
To reference this document use:
http://resolver.tudelft.nl/uuid:e5885071-7823-4eec-837a-51138ba8c333
DOI
https://doi.org/10.1016/j.reprotox.2008.12.009
TNO identifier
241460
ISSN
0890-6238
Source
Reproductive Toxicology, 27 (2), 93-102
Document type
article