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By - Agapios Sachinidis (University of Cologne)
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Agapios Sachinidis (University of Cologne):
Application of the embryonic stem cell model for drug discovery and development – a toxicogenomic approach
in vitro
bFGF
Generation of organ-specific cell types can be recapitulated under in vitro conditions
Advantage of in vitro human embryonic stem cell-based toxicity models
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N
O
F
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embryonic stem cell-based toxicity(reduce costs and time)
Highly automated, inexpensive Very expensive Most expensive
Basic Research
Discovery Development Commercial
TargetIdentification
TargetValidation
LeadIdentification
LeadOptimization Pre-clinical
ClinicalTrials
Post MarketTrials
TargetValidation
Enter of 5,000 compounds5 compounds
1 (Approval for therapeutic use)
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
100M 30M 10M 3M
Untreated
Toxicity of Cytarabine and Thalidomide in human EBs
ES cells-based toxicogenomics signatures
EB Control Cytarabine Thalidomide
Day 7
Day 14
RNA
EB Control Cytarabine Thalidomideundifferentiated
Microarray analysis
45000 transcripts
Randomly differentiated hESC (H9) treated with Thalidomide (IC10=10 µM) and Cytarabine (IC10=1 nM)
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
0 5 10 15 20 25
anatomical structuredevelopment
developmental process
metabolic process
multicellular organismaldevelopment
RNA metabolic process
regulation of transcription
anatomical structuremorphogenesis
regulation of geneexpression
Number of transcripts
Upregulated
0 10 20 30 40 50 60 70 80 90
developmental process
lipoprotein metabolic process
anatomical structure morphogenesis
coagulation
response to wounding
anatomical structure development
circulatory system process
system development
organ development
regulation of blood pressure
steroid metabolic process
lipid metabolic process
blood circulation
hemostasis
blood coagulation
digestion
Number of transcripts
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
DifferentiallyExpressed: 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
0 5 10 15 20 25 30 35 40
multicellular organismal development
neurogenesis
cellular developmental process
anatomical structure development
neurite morphogenesis
central nervous system development
axonogenesis
generation of neurons
neuron migration
nervous system development
developmental process
neuron morphogenesis duringdifferentiation
neuron differentiation
Number of transcripts
0 20 40 60 80 100 120
kidney development
developmental process
multicellular organismal process
metanephros development
urogenital system development
ossification
skeletal development
multicellular organismal development
anatomical structure development
organ development
heart development
mesoderm development
cartilage development
Number of transcripts
Downregulated
Immunocytochemistry for Cytarabine treated EBs on day 14 for the neuronal marker TUBB3 and Map2
Control1 nM Cytarabine
Morphology of hESC derived EBs (day14) on treatment with different concentrations of Cytarabine
1 Map2
TUBB3
TUBB3
TUBB3
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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
Control1 nM Cytarabine
Relative mRNA expression levels in Thalidomide treated EBs on day 14 compared with untreated day 14 EBs for mesodermal markers
Thalidomide FGB
-3.5
-3
-2.5
-2
-1.5
-1
-0.5
0
3 µM 10 µM 30 µM
Fo
ld C
ha
ng
e (
log
2)
Thalidomide, FGA
-3.5
-3
-2.5
-2
-1.5
-1
-0.5
0
3 µM 10 µM 30 µM
Fo
ld C
ha
ng
e (
log
2)
Thalidomide, PITX2
-1.8
-1.6
-1.4
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
3 µM 10 µM 30 µM
Fo
ld C
han
ge
(lo
g2)
Thalidomide (ANGPTL2)
-2.5
-2
-1.5
-1
-0.5
0
3 µM 10 µM 30 µM
Fo
ld C
ha
ng
e (
log
2)
Relative mRNA expression levels in Thalidomide treated EBs on day 14 compared with untreated day 14 EBs for endodermal marker AFP
-ActinAFP
ES
Co
ntr
ol
3 µ
M
10 µ
M
30 µ
M
Thalidomide, AFP
-3.5
-3
-2.5
-2
-1.5
-1
-0.5
0
3 µM 10 µM 30 µM
Fo
ld C
han
ge
(lo
g 2
)
Thalidomide, AFP
-3.5
-3
-2.5
-2
-1.5
-1
-0.5
0
3 µM 10 µM 30 µM
Fo
ld C
han
ge
(lo
g 2
)
Western blot validation for Cytarabine treated EBs on day 14 for neuronal marker TUBB3
-Actin
III Tubulin
ES
Co
ntr
ol
0.5n
M
1nM
Relative mRNA expression levels in Cytarabine treated EBs on day 14 compared with untreated day 14 EBs for neuronal markers.
Cytarabine, Pax7
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0.5 nM 1 nM 5 nM Sacharin
Fo
ld C
ha
ng
e (
log
2)
Cytarabine, Sox3
0
0.5
1
1.5
2
2.5
0.5 nM 1 nM 5 nM Sacharin
Fo
ld C
ha
ng
e (
log
2)
Cytarabin, Hoxa5
-1
0
1
2
3
4
5
6
0.5 nM 1 nM 5 nM Sacharin
Fo
ld C
ha
ng
e (
log
2)
Cytarabin, Nestin
-0.5
0
0.5
1
1.5
2
2.5
0.5 nM 1 nM 5 nM Sacharin
Fo
ld C
ha
ng
e (
log
2)
Cytarabin Map3
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0.5 nM 1 nM 5 nM Sacharin
Fo
ld C
ha
ng
e (
log
2)
