Assessment of technical protocols for novel embryonic stem cell tests with molecular markers (Hand1- and Cmya1-ESTs): a preliminary cross-laboratory performance analysis

Suzuki, Noriyuki; Yamashita, Norihisa; Koseki, Naoteru; Yamada, Tōru; Kimura, Yutaka; Aiba, Setsuya; Toyoizumi, Tomoyasu; Watanabe, Mika; Ohta, Ryo; Tanaka, Noriho; Saito, Koichi

doi:10.2131/jts.37.845

Cited by 15 publications

(6 citation statements)

References 17 publications

(17 reference statements)

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“…Additionally, different cell lines such as H1, SA002 and ReliCell hES1 were used for other investigations (Adler et al , , ; Mehta et al , ). However, we observed the tendency that a IC 50 value for 5‐fluorouracil or indomethacin is similar to recent research by other groups using with mES and hES cells in ESTs methods (Genschow et al , ; Mehta et al , ; Suzuki et al , ). These results suggest that hES cells and the conditions of our experiments were appropriate for assessing the IC 50 values of embryotoxic chemicals within a relatively short period.…”

Section: Discussionsupporting

confidence: 92%

“…Diversity related genetic factors, DNA methylation and DNA repair processes between experimental animals and humans have been reported in the development stage (Krtolica et al , ). Although EST using mES cells has routinely been used to evaluate embryotoxic chemicals (Spielmann et al , ; Suzuki et al , ), it has limits in incorrect classification of embryotoxic chemicals owing to species‐specific differences between humans and animals (Ginis et al , ).…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of developmental toxicity using undifferentiated human embryonic stem cells

Jung

Choi

Kang

et al. 2014

J of Applied Toxicology

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An embryonic stem cell test (EST) has been developed to evaluate the embryotoxic potential of chemicals with an in vitro system. In the present study, novel methods to screen toxic chemicals during the developmental process were evaluated using undifferentiated human embryonic stem (hES) cells. By using surface marker antigens (SSEA-4, TRA-1-60 and TRA-1-81), we confirmed undifferentiated conditions of the used hES cells by immunocytochemistry. We assessed the developmental toxicity of embryotoxic chemicals, 5-fluorouracil, indomethacin and non-embryotoxic penicillin G in different concentrations for up to 7 days. While expressions of the surface markers were not significantly affected, the embryotoxic chemicals influenced their response to pluripotent ES cell markers, such as OCT-4, NANOG, endothelin receptor type B (EDNRB), secreted frizzled related protein 2 (SFRP2), teratocarcinoma-derived growth factor 1 (TDGF1), and phosphatase and tensin homolog (PTEN). Most of the pluripotent ES cell markers were down-regulated in a dose-dependent manner after treatment with embryotoxic chemicals. After treatment with 5-fluorouracil, indomethacin and penicillin G, we observed a remarkable convergence in the degree of up-regulation of development, cell cycle and apoptosis-related genes by gene expression profiles using an Affymetrix GeneChips. Taken together, these results suggest that embryotoxic chemicals have cytotoxic effects, and modulate the expression of ES cell markers as well as development-, cell cycle- and apoptosis-related genes that have pivotal roles in undifferentiated hES cells. Therefore, we suggest that hES cells may be useful for testing the toxic effects of chemicals that could impact the embryonic developmental stage.

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Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Evaluation of developmental toxicity using undifferentiated human embryonic stem cells

Jung

Choi

Kang

et al. 2014

J of Applied Toxicology

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“…While undifferentiated mouse ES cells have been used as ESTs, we evaluated embryotoxic chemicals in the vitro system using undifferentiated human ES cells because use of mouse ES cells has reached its limits due to interspecies distinction (53). When we modified the protocol for measurement of IC 50 values after exposure of embryotoxic chemicals for up to seven days, the IC 50 values of embryotoxic chemicals were similar to those reported by other groups using ESTs with mouse ES (14) and human ES cells (18,54). This result suggests that our system was appropriate for assessment of the IC 50 values of embryotoxic chemicals within a relatively short period.…”

Section: Developmental Toxicants On Undifferentiated Human Es Cellssupporting

confidence: 63%

Assessment of Developmental Toxicants using Human Embryonic Stem Cells

Hong¹,

Jeung²

2013

Toxicological Research

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Embryonic stem (ES) cells have potential for use in evaluation of developmental toxicity because they are generated in large numbers and differentiate into three germ layers following formation of embryoid bodies (EBs). In earlier study, embryonic stem cell test (EST) was established for assessment of the embryotoxic potential of compounds. Using EBs indicating the onset of differentiation of mouse ES cells, many toxicologists have refined the developmental toxicity of a variety of compounds. However, due to some limitation of the EST method resulting from species-specific differences between humans and mouse, it is an incomplete approach. In this regard, we examined the effects of several developmental toxic chemicals on formation of EBs using human ES cells. Although human ES cells are fastidious in culture and differentiation, we concluded that the relevancy of our experimental method is more accurate than that of EST using mouse ES cells. These types of studies could extend our understanding of how human ES cells could be used for monitoring developmental toxicity and its relevance in relation to its differentiation progress. In addition, this concept will be used as a model system for screening for developmental toxicity of various chemicals. This article might update new information about the usage of embryonic stem cells in the context of their possible ability in the toxicological fields.

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“…The conclusion of the study was high predictability and accuracy with reduced test duration and manpower compared to the original EST. Furthermore, in 2012, we published another study (Suzuki et al, 2012) in which we investigated Hand1-and Cmya1-ESTs to explore reproducibility by comparing a set of 6 well-known test chemicals, including hydroxyurea, at four different laboratories. We managed to obtain good correspondence among all four laboratories, proving the high potency of transferability, intra-laboratory variability, and inter-laboratory variability of the protocols.…”

Section: Introductionmentioning

confidence: 99%

Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST): A novel rapid and highly reproducible <i>in vitro</i> test for embryotoxicity by measuring cytotoxicity and differentiation toxicity using engineered mouse ES cells

et al. 2015

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-The embryonic stem cell test (EST) is a promising alternative method for evaluating embryotoxicity of test chemicals by measuring cytotoxicity and differentiation toxicity using mouse ES cells. Differentiation toxicity is analyzed by microscopically counting the beating of embryonic bodies after 10 days of culture. However, improvements are necessary to reduce the laborious manipulations involved and the time required to obtain results. We have previously reported the successful stable transfection of ES cells (ES-D3) with the heart and neural crest derivatives expressed transcript 1 (Hand1) gene and the establishment of a 96-well multi-plate-based new EST with luciferase reporter assay 6 days after treatment with test chemicals. Now, we propose an even more rapid and easier EST, named Hand1-Luc EST. We established another cell line to monitor the Hand1 gene expression via a luciferase reporter gene. By mRNA analysis and luciferase assay, we examined in detail the luciferase activity during cell differentiation, which allowed us to reduce the time of measurement from day 6 to day 5 (120 hr). Furthermore, the protocol was improved, with, among others, the measurement of cytotoxicity and differentiation toxicity taking place in the same 96-well round bottom plate instead of two different plates. With the positive control, 5-fluorouracil (5-FU), and 9 test chemicals, data with high reproducibility and very low variation (CV < 50%) in the relevant endpoints were obtained. This study shows that the Hand1-Luc EST could provide an accurate and sensitive short-term test for prediction of embryotoxicants by measuring cytotoxicity and differentiation toxicity from the same sample.

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Assessment of technical protocols for novel embryonic stem cell tests with molecular markers (Hand1- and Cmya1-ESTs): a preliminary cross-laboratory performance analysis

Cited by 15 publications

References 17 publications

Evaluation of developmental toxicity using undifferentiated human embryonic stem cells

Evaluation of developmental toxicity using undifferentiated human embryonic stem cells

Assessment of Developmental Toxicants using Human Embryonic Stem Cells

Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST): A novel rapid and highly reproducible <i>in vitro</i> test for embryotoxicity by measuring cytotoxicity and differentiation toxicity using engineered mouse ES cells

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