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Application of the embryonic stem cell model for drug discovery and development – a toxicogenomic approach
 

Application of the embryonic stem cell model for drug discovery and development – a toxicogenomic approach

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By - Agapios Sachinidis (University of Cologne)

By - Agapios Sachinidis (University of Cologne)

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    Application of the embryonic stem cell model for drug discovery and development – a toxicogenomic approach Application of the embryonic stem cell model for drug discovery and development – a toxicogenomic approach Presentation Transcript

    • Agapios Sachinidis (University of Cologne): Application of the embryonic stem cell model for drug discovery and development – a toxicogenomic approach
    • Generation of organ-specific cell types can be recapitulated under in vitro conditions in vitro bFGF
    • Advantage of in vitro human embryonic stem cell-based toxicity models embryonic stem cell-based toxicity (reduce costs and time) Highly automated, inexpensive Very expensive Most expensive Basic Research Discovery Development Commercial Target Identification Target Validation Lead Identification Lead Optimization Pre - clinical Clinical Trials Post Market Trials Target Validation Enter of 5,000 compounds 5 compounds 1 (Approval for therapeutic use)
    • Toxicity of Cytarabine and Thalidomide in human EBs Coated with 5 % pluronic H9 clumps added (30 clumps / well) Aggregation Culture till 3-4 days on the plate Transferred to bacteriological plate with differentiation medium grown till day 7 or 14 Method for EB formation: Use 96-well plate Shaking +drugs CY TH 100nM 30nM 10nM 1nM 100  M 30  M 10  M 3  M Untreated
    • Randomly differentiated hESC (H9) treated with Thalidomide (IC10=10 µM) and Cytarabine (IC10=1 nM) ES cells-based toxicogenomics signatures EB Control Cytarabine Thalidomide Day 7 Day 14 RNA EB Control Cytarabine Thalidomide undifferentiated Microarray analysis 45000 transcripts
    • Gene expression profiling of human embryonic stem cells exposed to Thalidomide Thalidomide treated (14 - days EBs ) vs untreated (14 - days EBs ) Differential expressed : 1134 transcripts Upregulated : 451 Downregulated : 683 Fold change (FC) value : ≥ 2, 45 transcripts ≥ -2, 144 transcripts Differentially Expressed transcripts
    • Gene Ontology analysis of the upregulated and downregulated transcripts in thalidomide -treated 14-days EBs compared to untreated 14-days EB Upregulated Downregulated
    • Gene expression profiling of human embryonic stem cells exposed to Cytarabine Differentially expressed transcripts Cytarabine-treated 14 -days EBs vs untreated 14- days EBs Differentially Expressed : 2732 transcripts Upregulated : 1307 transcripts Downregulated : 1424 transcripts Fold change value : ≥ 2, 166 tranascripts ≥ -2, 398 tranascripts
    • Gene Ontology analysis of the upregulated and downregulated transcripts in Cytarabine-treated 14-days EBs compared to untreated 14-days EB Upregulated Downregulated
    • Immunocytochemistry for Cytarabine treated EBs on day 14 for the neuronal marker TUBB3 and Map2 Control 1 nM Cytarabine Morphology of hESC derived EBs (day14) on treatment with different concentrations of Cytarabine 1 Map2 TUBB3 TUBB3 TUBB3
    • Summary and conclusions
      • Thalidomide and Cytarabine-treated differentiating human ES cells showed abnormal expression of several developmental genes that can be used as developmental toxicity markers
      • Cytarabine-treated differentiated hES cells showed a significant upregulation of transcripts involved in neuronal development
      • ES cells in combination with -omics technologies potentially offer a good in vitro toxicity model for identification of biomarkers (genes, phosphoproteins etc.) and might contribute to an effective drug development
    • Thank You
    •  
    • Real time, Immunohistochemistry and Western-blotting protein validation also prove the microarray data
    • Immunocytochemistry for Cytarabine treated EBs on day 14 for neuronal marker Map2 Control 1 nM Cytarabine
    • Relative mRNA expression levels in Thalidomide treated EBs on day 14 compared with untreated day 14 EBs for mesodermal markers
    • Relative mRNA expression levels in Thalidomide treated EBs on day 14 compared with untreated day 14 EBs for endodermal marker AFP  -Actin AFP ES Control 3 µM 10 µM 30 µM
    • Western blot validation for Cytarabine treated EBs on day 14 for neuronal marker TUBB3  -Actin  III Tubulin ES Control 0.5nM 1nM
    • Relative mRNA expression levels in Cytarabine treated EBs on day 14 compared with untreated day 14 EBs for neuronal markers.