Publications (5)22.77 Total impact
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Article: Testing potential developmental toxicants with a cytotoxicity assay based on human embryonic stem cells.
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ABSTRACT: Since the differentiation of embryonic stem cells mimics early development, these cells could potentially permit the detection of embryotoxicants which interfere with this process. Although reliable tests based on murine embryonic stem cells exist, no such methods are available for human embryonic stem (hES) cells. Nonetheless, to avoid the false classification of substances due to inter-species differences, human-relevant toxicity tests are needed. We therefore developed an assay based on three human cell types, representing different degrees of developmental maturation, namely, human foreskin fibroblasts, hES cell-derived progenitor cells, and pluripotent hES cells. A set of embryotoxicants for which existing in vivo data were available, namely, all-trans retinoic acid (ATRA), 13-cis retinoic acid (13CRA), valproic acid (VPA) and dimethyl sulphoxide (DMSO), were tested. 5-fluorouracil (5-FU) was used as a positive control, and saccharin as a negative control. Two methods were compared for the assessment of cell viability -- the determination of intracellular ATP content and of resazurin reduction. In addition, the protective capacity of basic fibroblast growth factor (bFGF) against retinoid-induced toxicity was investigated. This novel assay system reliably detected the embryotoxic potentials of the test substances, 5-FU, ATRA, 13-CRA (a substance that displays inter-species differences in its effects) and VPA. This was possible due to the apparent differences in the sensitivities of the human cell types used in the assay system. Thus, our results clearly indicate the advantages and relevance of using hES cells in in vitro developmental toxicity testing.Alternatives to laboratory animals: ATLA 05/2008; 36(2):129-40. · 1.58 Impact Factor -
Article: Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture.
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ABSTRACT: There have been recent reports of human embryonic stem cell (hESC) lines developing chromosomal aberrations after long-term culture, indicating an unstable genomic status due to the in vitro milieu. This raises concern, since it would limit their use in therapeutics. In this study the chromosomal status of five well-characterized hESC lines, SA002, SA002.5, AS034.1.1, SA121 and SA461, was monitored during long-term in vitro culture. The criteria of defined hESCs were met by all of the five hESC lines (four diploid and one trisomic for chromosome 13). The genomes were screened for chromosomal aberrations and rearrangements using comparative genomic hybridization (CGH), interphase fluorescence in situ hybridization (FISH) and traditional karyotyping on several occasions while in culture. The genomic integrity was shown to be maintained after repeated freeze-thaw procedures and continuous culture in vitro for up to 22 months (148 passages). We discuss the most common de novo chromosomal aberrations reported in hESCs, as well as their possible origin.Chromosome Research 02/2006; 14(2):131-7. · 3.09 Impact Factor -
Article: Large-scale propagation of four undifferentiated human embryonic stem cell lines in a feeder-free culture system.
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ABSTRACT: We describe an improved and more robust protocol for transfer and subsequent propagation of human embryonic stem cells under feeder-free conditions. The results show that mechanical dissociation for transfer of the human embryonic stem cells to Matrigel resulted in highest survival rates. For passage of the cultures on the other hand, enzymatic dissociation was found to be most efficient. In addition, this method reduces the time, work, and skills needed for propagation of the human embryonic stem cells. With the present protocol, the human embryonic stem cells have been cultured under feeder-free conditions for up to 35 passages while maintaining a normal karyotype, stable proliferation rate, and high telomerase activity. Furthermore, the feeder-free human embryonic stem cell cultures express the transcription factor Oct-4, alkaline phosphatase, and cell surface markers SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, and formed teratomas in severe combined immunodeficient mice. This method provides distinct advantages compared with previous protocols and make propagation of human embryonic stem cells less laborious and more efficient.Developmental Dynamics 09/2005; 233(4):1304-14. · 2.54 Impact Factor -
Article: High-resolution analysis of the subtelomeric regions of human embryonic stem cells.
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ABSTRACT: The use of human embryonic stem cells (hESCs) in most applications is dependent on their undifferentiated proliferation in vitro. Recent studies have illustrated the possibility that chromosomal changes may occur in hESCs during in vitro propagation of these cells. However, no studies so far have screened for chromosomal abnormalities in hESCs using high-resolution techniques that can detect alterations on a few base-pair levels. We have used the recently developed multiplex ligation-dependent probe amplification procedure to analyze the possible occurrence of deletions or duplications in the subtelomeric regions of hESCs in early and late passages. In this study we show that no subtelomeric anomalies were detected in any of the nine hESC lines investigated, supporting the conclusion that hESCs, under appropriate conditions, maintain genomic stability during in vitro propagation.Stem Cells 05/2005; 23(4):483-8. · 7.78 Impact Factor -
Article: High‐Resolution Analysis of the Subtelomeric Regions of Human Embryonic Stem Cells
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ABSTRACT: The use of human embryonic stem cells (hESCs) in most applications is dependent on their undifferentiated proliferation in vitro. Recent studies have illustrated the possibility that chromosomal changes may occur in hESCs during in vitro propagation of these cells. However, no studies so far have screened for chromosomal abnormalities in hESCs using high-resolution techniques that can detect alterations on a few base-pair levels. We have used the recently developed multiplex ligation-dependent probe amplification procedure to analyze the possible occurrence of deletions or duplications in the subtelomeric regions of hESCs in early and late passages. In this study we show that no subtelomeric anomalies were detected in any of the nine hESC lines investigated, supporting the conclusion that hESCs, under appropriate conditions, maintain genomic stability during in vitro propagation.Stem Cells 03/2005; 23(4):483 - 488. · 7.78 Impact Factor
