Science & Insights

@article{Shinde2016b,

title = {Comparison of a teratogenic transcriptome-based predictive test based on human embryonic versus inducible pluripotent stem cells},

author = {Vaibhav Shinde and Sureshkumar Perumal Srinivasan and Margit Henry and Tamara Rotshteyn and Jürgen Hescheler and Jörg Rahnenführer and Marianna Grinberg and Johannes Meisig and Nils Blüthgen and Tanja Waldmann and Marcel Leist and Jan G. Hengstler and Agapios Sachinidis},

url = {https://trenzyme.com/cell-culture-services/stem-cell-differentiation-service/, ➥Stem Cell Differentiation Service},

doi = {10.1186/s13287-016-0449-2},

issn = {1757-6512},

year  = {2016},

date = {2016-12-00},

urldate = {2016-12-00},

journal = {Stem Cell Res Ther},

volume = {7},

number = {1},

publisher = {Springer Science and Business Media LLC},

abstract = {Background

Human embryonic stem cells (hESCs) partially recapitulate early embryonic three germ layer development, allowing testing of potential teratogenic hazards. Because use of hESCs is ethically debated, we investigated the potential for human induced pluripotent stem cells (hiPSCs) to replace hESCs in such tests.



Methods

Three cell lines, comprising hiPSCs (foreskin and IMR90) and hESCs (H9) were differentiated for 14 days. Their transcriptome profiles were obtained on day 0 and day 14 and analyzed by comprehensive bioinformatics tools.



Results

The transcriptomes on day 14 showed that more than 70% of the “developmental genes” (regulated genes with > 2-fold change on day 14 compared to day 0) exhibited variability among cell lines. The developmental genes belonging to all three cell lines captured biological processes and KEGG pathways related to all three germ layer embryonic development. In addition, transcriptome profiles were obtained after 14 days of exposure to teratogenic valproic acid (VPA) during differentiation. Although the differentially regulated genes between treated and untreated samples showed more than 90% variability among cell lines, VPA clearly antagonized the expression of developmental genes in all cell lines: suppressing upregulated developmental genes, while inducing downregulated ones. To quantify VPA-disturbed development based on developmental genes, we estimated the “developmental potency” (D p) and “developmental index” (D i).



Conclusions

Despite differences in genes deregulated by VPA, uniform D i values were obtained for all three cell lines. Given that the D i values for VPA were similar for hESCs and hiPSCs, D i can be used for robust hazard identification, irrespective of whether hESCs or hiPSCs are used in the test systems.},

keywords = {iPSC},

pubstate = {published},

tppubtype = {article}

}

@article{Smirnova2015,

title = {A LUHMES 3D dopaminergic neuronal model for neurotoxicity testing allowing long-term exposure and cellular resilience analysis},

author = {Lena Smirnova and G. Harris and Johannes Delp and M. Valadares and D. Pamies and Helena T. Hogberg and Tanja Waldmann and Marcel Leist and Thomas Hartung},

url = {https://trenzyme.com/cell-culture-services/stem-cell-differentiation-service/, ➥Stem Cell Differentiation Service},

doi = {10.1007/s00204-015-1637-z},

issn = {1432-0738},

year  = {2016},

date = {2016-11-00},

urldate = {2016-11-00},

journal = {Arch Toxicol},

volume = {90},

number = {11},

pages = {2725--2743},

publisher = {Springer Science and Business Media LLC},

abstract = {Several shortcomings of current Parkinson’s disease (PD) models limit progress in identification of environmental contributions to disease pathogenesis. The conditionally immortalized cell line LUHMES promises to make human dopaminergic neuronal cultures more easily available, but these cells are difficult to culture for extended periods of time. We overcame this problem by culturing them in 3D with minor medium modifications. The 3D neuronal aggregates allowed penetration by small molecules and sufficient oxygen and nutrient supply for survival of the innermost cells. Using confocal microscopy, gene expression, and flow cytometry, we characterized the 3D model and observed a highly reproducible differentiation process. Visualization and quantification of neurites in aggregates was achieved by adding 2 % red fluorescent protein-transfected LUHMES cells. The mitochondrial toxicants and established experimental PD agents, rotenone and MPP+, perturbed genes involved in one-carbon metabolism and transsulfuration pathways (ASS1, CTH, and SHTM2) as in 2D cultures. We showed, for the first time in LUHMES, down-regulation of mir-7, a miRNA known to target alpha-synuclein and to be involved in PD. This was observed as early as 12 h after rotenone exposure, when pro-apoptotic mir-16 and rotenone-sensitive mir-210 were not yet significantly perturbed. Finally, washout experiments demonstrated that withdrawal of rotenone led to counter-regulation of mir-7 and ASS1, CTH, and SHTM2 genes. This suggests a possible role of these genes in direct cellular response to the toxicant, and the model appears to be suitable to address the processes of resilience and recovery in neurotoxicology and Parkinson’s disease in future studies.},

keywords = {LUHMES},

pubstate = {published},

tppubtype = {article}

}

@article{Hoelting2016,

title = {Stem Cell-Derived Immature Human Dorsal Root Ganglia Neurons to Identify Peripheral Neurotoxicants},

author = {Lisa Hoelting and Stefanie Klima and Christiaan Karreman and Marianna Grinberg and Johannes Meisig and Margit Henry and Tamara Rotshteyn and Jörg Rahnenführer and Nils Blüthgen and Agapios Sachinidis and Tanja Waldmann and Marcel Leist},

url = {https://trenzyme.com/cell-culture-services/stem-cell-differentiation-service/, ➥Stem Cell Differentiation Service},

doi = {10.5966/sctm.2015-0108},

issn = {2157-6580},

year  = {2016},

date = {2016-04-01},

urldate = {2016-04-01},

journal = {Stem Cells Translational Medicine},

volume = {5},

number = {4},

pages = {476--487},

publisher = {Oxford University Press (OUP)},

abstract = {Safety sciences and the identification of chemical hazards have been seen as one of the most immediate practical applications of human pluripotent stem cell technology. Protocols for the generation of many desirable human cell types have been developed, but optimization of neuronal models for toxicological use has been astonishingly slow, and the wide, clinically important field of peripheral neurotoxicity is still largely unexplored. A two-step protocol to generate large lots of identical peripheral human neuronal precursors was characterized and adapted to the measurement of peripheral neurotoxicity. High content imaging allowed an unbiased assessment of cell morphology and viability. The computational quantification of neurite growth as a functional parameter highly sensitive to disturbances by toxicants was used as an endpoint reflecting specific neurotoxicity. The differentiation of cells toward dorsal root ganglia neurons was tracked in relation to a large background data set based on gene expression microarrays. On this basis, a peripheral neurotoxicity (PeriTox) test was developed as a first toxicological assay that harnesses the potential of human pluripotent stem cells to generate cell types/tissues that are not otherwise available for the prediction of human systemic organ toxicity. Testing of more than 30 chemicals showed that human neurotoxicants and neurite growth enhancers were correctly identified. Various classes of chemotherapeutic agents causing human peripheral neuropathies were identified, and they were missed when tested on human central neurons. The PeriTox test we established shows the potential of human stem cells for clinically relevant safety testing of drugs in use and of new emerging candidates.},

keywords = {PeriTox},

pubstate = {published},

tppubtype = {article}

}

@article{Pallocca2015,

title = {Identification of transcriptome signatures and biomarkers specific for potential developmental toxicants inhibiting human neural crest cell migration},

author = {Giorgia Pallocca and Marianna Grinberg and Margit Henry and Tancred Frickey and Jan G. Hengstler and Tanja Waldmann and Agapios Sachinidis and Jörg Rahnenführer and Marcel Leist},

url = {https://trenzyme.com/cell-culture-services/cell-based-assay-development-service/, ➥Cell Based Development Service},

doi = {10.1007/s00204-015-1658-7},

issn = {1432-0738},

year  = {2016},

date = {2016-01-00},

urldate = {2016-01-00},

journal = {Arch Toxicol},

volume = {90},

number = {1},

pages = {159--180},

publisher = {Springer Science and Business Media LLC},

abstract = {The in vitro test battery of the European research consortium ESNATS (‘novel stem cell-based test systems’) has been used to screen for potential human developmental toxicants. As part of this effort, the migration of neural crest (MINC) assay has been used to evaluate chemical effects on neural crest function. It identified some drug-like compounds in addition to known environmental toxicants. The hits included the HSP90 inhibitor geldanamycin, the chemotherapeutic arsenic trioxide, the flame-retardant PBDE-99, the pesticide triadimefon and the histone deacetylase inhibitors valproic acid and trichostatin A. Transcriptome changes triggered by these substances in human neural crest cells were recorded and analysed here to answer three questions: (1) can toxicants be individually identified based on their transcript profile; (2) how can the toxicity pattern reflected by transcript changes be compacted/dimensionality-reduced for practical regulatory use; (3) how can a reduced set of biomarkers be selected for large-scale follow-up? Transcript profiling allowed clear separation of different toxicants and the identification of toxicant types in a blinded test study. We also developed a diagrammatic system to visualize and compare toxicity patterns of a group of chemicals by giving a quantitative overview of altered superordinate biological processes (e.g. activation of KEGG pathways or overrepresentation of gene ontology terms). The transcript data were mined for potential markers of toxicity, and 39 transcripts were selected to either indicate general developmental toxicity or distinguish compounds with different modes-of-action in read-across. In summary, we found inclusion of transcriptome data to largely increase the information from the MINC phenotypic test.},

keywords = {MINC},

pubstate = {published},

tppubtype = {article}

}

@article{Dreser2015,

title = {Grouping of histone deacetylase inhibitors and other toxicants disturbing neural crest migration by transcriptional profiling},

author = {Nadine Dreser and Bastian Zimmer and Christian Dietz and Elena Sügis and Giorgia Pallocca and Johanna Nyffeler and Johannes Meisig and Nils Blüthgen and Michael R. Berthold and Tanja Waldmann and Marcel Leist},

url = {https://trenzyme.com/cell-culture-services/cell-based-assay-development-service/, ➥Cell Based Development Service},

doi = {10.1016/j.neuro.2015.07.008},

issn = {0161-813X},

year  = {2015},

date = {2015-09-00},

urldate = {2015-09-00},

journal = {NeuroToxicology},

volume = {50},

pages = {56--70},

publisher = {Elsevier BV},

abstract = {Functional assays, such as the “migration inhibition of neural crest cells” (MINC) developmental toxicity test, can identify toxicants without requiring knowledge on their mode of action (MoA). Here, we were interested, whether (i) inhibition of migration by structurally diverse toxicants resulted in a unified signature of transcriptional changes; (ii) whether statistically-identified transcript patterns would inform on compound grouping even though individual genes were little regulated, and (iii) whether analysis of a small group of biologically-relevant transcripts would allow the grouping of compounds according to their MoA. We analyzed transcripts of 35 ‘migration genes’ after treatment with 16 migration-inhibiting toxicants. Clustering, principal component analysis and correlation analyses of the data showed that mechanistically related compounds (e.g. histone deacetylase inhibitors (HDACi), PCBs) triggered similar transcriptional changes, but groups of structurally diverse toxicants largely differed in their transcriptional effects. Linear discriminant analysis (LDA) confirmed the specific clustering of HDACi across multiple separate experiments. Similarity of the signatures of the HDACi trichostatin A and suberoylanilide hydroxamic acid to the one of valproic acid (VPA), suggested that the latter compound acts as HDACi when impairing neural crest migration. In conclusion, the data suggest that (i) a given functional effect (e.g. inhibition of migration) can be associated with highly diverse signatures of transcript changes; (ii) statistically significant grouping of mechanistically-related compounds can be achieved on the basis of few genes with small regulations. Thus, incorporation of mechanistic markers in functional in vitro tests may support read-across procedures, also for structurally un-related compounds.},

keywords = {MINC},

pubstate = {published},

tppubtype = {article}

}