Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies: present state and future prospects

Rohwedel, Jürgen; Guan, Kaomei; Hegert, Claudia; Wobus, Anna M.

doi:10.1016/s0887-2333(01)00074-1

Cited by 141 publications

(80 citation statements)

References 97 publications

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“…In recent years, human embryonic stem (hES) cells have generated much interest over their potential applications in clinical therapy [1], basic scientific research [2,3] and for pharmacological and cytotoxicity screening in vitro [4,5]. Nevertheless, a major bottleneck impeding the widespread application of these cells is the lack of efficient cryopreservation protocols.…”

Section: Introductionmentioning

confidence: 99%

Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis

et al. 2005

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SummaryA major challenge in the widespread application of human embryonic stem (hES) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of <5% as reported by previous studies. This study characterized cell death within frozen-thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (%98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37°C incubator, there was observed to be a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed-down by keeping the freshly-thawed hES colonies at 4°C, with >90% of cells remaining viable after 90 min of incubation at 4°C. This effect was reversible upon re-exposing the cells to physiological temperature. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37°C. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay confirmed apoptosis-induced nuclear DNA fragmentation in frozen-thawed hES cells after incubation at 37°C for 90 min. Expression of active caspase-3 enzyme, which is another prominent marker of apoptosis, was confirmed by immunocytochemical staining, while transmission electron microscopy showed typical ultrastructural features of apoptosis such as chromatin condensation and margination to the nuclear membrane. Hence, our results demonstrated that apoptosis instead of cellular necrosis, is the major mechanism of the loss of viability of cryopreserved hES cells during freeze-thawing with conventional slow-cooling protocols.

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Section: Introductionmentioning

confidence: 99%

Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis

et al. 2005

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“…ES cells are unique in their capacity to form all cells of the developing embryo and are useful in studies ranging from in vitro differentiation to knockout mice [2][3][4][5][6][7]. Therefore, ES cells are an important tool for both basic and applied research, and the efficient manipulation of these cells is critical for their use in such studies.…”

Section: Introductionmentioning

confidence: 99%

The Human Cytomegalovirus Immediate‐Early Promoter is Transcriptionally Active in Undifferentiated Mouse Embryonic Stem Cells

Ward

Stern

2002

STEM CELLS

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It has been reported recently that the cytomegalovirus (CMV) immediate-early promoter is transcriptionally inactive in undifferentiated mouse embryonic stem (ES) cells. This result is surprising, since the CMV promoter is used to express transgenes in a variety of cell lines. We studied the expression of a human CMV-driven enhanced green fluorescent protein (EGFP) reporter gene (pEGFP-N1) in five undifferentiated mouse ES cell lines (BL/6III, D3, E14TG2a, MESC20, and 129) and found EGFP expression in all of these cell lines. Under optimal conditions, between 50%-80% transfection efficiencies could be achieved, and EGFP expression levels were maintained for at least 72 hours. Therefore, the human CMV promoter remains a useful system for transgene expression in undifferentiated ES cells. Stem Cells 2002;20:472-475 STEM CELLS 2002;20:472-475 www.StemCells.com

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“…Available literature suggests that [6,32,45] the different types of stem cells are involved in toxicity testing and screening of organ-specific toxicity (Fig. 8).…”

Section: Fig 5: Es Cell Derived Differentiation-related Endpoints Usmentioning

confidence: 99%

Mesenchymal Stem Cells: An Innovative Approach in Pharmacokinetics

Tripathy¹,

Mohanty²

2017

Asian J Pharm Clin Res

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Multipotent mesenchymal stem cells (MSCs) are special kind of stem cells which originate from mesenchyme. These cells can be used as an imperative tool to study reproductive toxicity, carcinogenicity, mutagenicity, genotoxicity, and pharmacokinetics. This novel system may reveal toxicantinduced etiology, decipher detailed understanding on molecular mechanisms of toxicants induced pathways and also enumerate the safe dose of an investigational product. Hence, this could ultimately replace, improve or overtake current predictive models in toxicology. The particular review describes the utilization of MSCs in different field of toxicological and pharmacological research.

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Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies: present state and future prospects

Cited by 141 publications

References 97 publications

Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis

Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis

The Human Cytomegalovirus Immediate‐Early Promoter is Transcriptionally Active in Undifferentiated Mouse Embryonic Stem Cells

Mesenchymal Stem Cells: An Innovative Approach in Pharmacokinetics

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