By - Agapios Sachinidis (University of Cologne)

1. Agapios Sachinidis (University of Cologne): Application of the embryonic stem cell model for drug discovery and development – a toxicogenomic approach

2. Generation of organ-specific cell types can be recapitulated under in vitro conditions in vitro bFGF

3. 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)

4. 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

5. 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

6. 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

7. Gene Ontology analysis of the upregulated and downregulated transcripts in thalidomide -treated 14-days EBs compared to untreated 14-days EB Upregulated Downregulated

8. 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

9. Gene Ontology analysis of the upregulated and downregulated transcripts in Cytarabine-treated 14-days EBs compared to untreated 14-days EB Upregulated Downregulated

10. 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

12. Thank You

14. Real time, Immunohistochemistry and Western-blotting protein validation also prove the microarray data

15. Immunocytochemistry for Cytarabine treated EBs on day 14 for neuronal marker Map2 Control 1 nM Cytarabine

16. Relative mRNA expression levels in Thalidomide treated EBs on day 14 compared with untreated day 14 EBs for mesodermal markers

17. 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

18. Western blot validation for Cytarabine treated EBs on day 14 for neuronal marker TUBB3  -Actin  III Tubulin ES Control 0.5nM 1nM

19. Relative mRNA expression levels in Cytarabine treated EBs on day 14 compared with untreated day 14 EBs for neuronal markers.