Human embryonic stem cells for in-vitro developmental toxicity testing HESI workshop on alternatives assays for developmental toxicity
Raimund Strehl Cellartis – The company
• Founded in early 2001, University spin-off, 36 employees • Focus on human embryonic stem cells for in-vitro drug discovery and toxicology
• Operating on a high quality standard • Academic & Industry collaborations 150+ • Major research grants: NIH - US$ 900 000 JDRF - US$ 600 000 BetaCellTherapy EU grants - € 3 million Vitrocellomics
HeartRepair
Stems
InvitroHeart
Carcinogenomics Cellartis - Facilities
Cellartis Sweden Cellartis UK
• ”Tailor-made” for hES cell work • Upscaling of hES cell production • Three cell culture laboratories that • Cell culture laboratories comply comply with GMP (EU standard) with GMP standard (3 EU standard + 1 US standard) • Operational in mid 2007 Human embryonic stem (hES) cell lines hES cell lines
• >30 Cellartis cell lines + collaborative lines (e.g. CECOL14 from Latin America)
• Two hES cell lines + subclones on the NIH stem cell registry
• Six hES cell lines for use in Germany
• Twenty hES cell lines in the UK stem cell registry
• First derivation of a truly xeno-free hES cell line
• Preparation and quality control of working cell banks (>100 vials) = LOT
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Ellerström et al., Stem Cells 2007 (provisionally accepted) Bibikova et al., Genome Research 2006 (in press) Ellerström et al., Stem Cells on line 2006 June 1 Caisander et al., Chrom Res 2006, 14:131-137 Heins et.al. J Biotechnol. 2005, 122:511-520 Maitra et al., Nature Genetics 2005, 37:1099-1103 Noaksson et al., Stem Cells 2005, 23:1460-1467 Darnfors et al., Stem Cells 2005, 23: 483-488 Sjögren et.al. Reprod Biomed Online. 2004, 9:326-329 Heins et. al. Stem Cells 2004, 22:367-376 hES cell lines - Establishment and expansion
Cellartis hES cell lines - LOT quality control
• Morphology • Thawing recovery rate of frozen LOT:s • Stem cell markers: SSEA-3, SSEA-4, SSEA-1, TRA-1-60, TRA-1-81, and Oct-4 • ALP- and Telomerase activity • Cytogenetic analysis (Karyotyping, FISH, MLPA) • In-vitro and in-vivo pluripotency • Mycoplasma and human viruses (Human Immunodeficiency Virus type 1 and 2, Hepatitis B and C, Cytomegalovirus, Herpes Simplex Virus type 1 and 2, Epstein-Barr Virus, Human Papilloma Virus) hES cell lines - Summary SA001, LOT AK001
Parameter Passage Result
LOT preparation p13 >100 vials Thawing recovery rate p13 - p14 100% SSEA-1 p31 Negative SSEA-3 p31 Positive SSEA-4 p31 Positive TRA-1-60 p31 Positive TRA-1-81 p31 Positive Oct-4 p31 Positive Alkaline phosphatase p31 Positive Karyotype p26, p27, p50 and p52 46, XY FISH (X, Y, 13, 18 and 21) p32 and p51 Diploid, XY MLPA (sub-telomeric regions) p26 No deletions Telomerase activity p27 Positive Pluripotency in vitro p29 Endo-, ecto-, mesoderm Pluripotency in vivo p19 Endo-, ecto-, mesoderm hES cell lines - Increasing demand for pluripotent hES cells
Production of pluripotent hES cells
Two shifts Scaleable culture methods Automated production / Reactors
Demand for pluripotent hES cells
Mostly for internal R+D Hepatocytes Cardiomyocytes Increased distribution to commercial collaborators
Mostly for products Hepatocytes Cardiomyocytes Screening hES cell lines – Overcoming culture challenges
Manual microdissection • Highest quality (Hand-picked) • Maintains normal lines
• Not scaleable • Laborios and expensive
Enzymatic propagation • Easier culture • More cells / dish • Scaleable • Relatively cheap • Automateable in the future hES cell lines – Robust Upscaling
Improved feeder-systems Single cell passaging
Flask / Multiwell culture Defined culture systems Application of hES cells in developmental toxicology Developmental toxicology - A humanized EST ?
A human developmental toxicity assay composed of two parts:
1. Cytotoxicity assays to detect a selective effect of substances on the viability of human embryonic cells.
2. hES cell based in-vitro development assay to evaluate influences on the mechanisms of human embryonic differentiation.
Cytotoxicity in embryonic cells Prediction Substance Mathematical of to be correlation developmental tested model Influence on toxicity differentiation Developmental toxicology - Differential cytotoxicity
Comparative cytotoxicity assays using 3 human cell types of increasing degree of maturation
Degree of maturation
Undifferentiated hES cell derived Human foreskin hES cells progenitors fibroblasts Developmental toxicology - Viability endpoint
Test protocol: Day -1: Seed cells Day 0: Start exposure Day 4: Change medium Day 7: Change medium Day 10: Analysis in plate reader
ATP content (Promega CellTiterGloTM) Resazurin reduction Developmental toxicology - Non-toxicant vs. Strong toxicant
5-FU Saccharin hFF hFF 150 150 Prog cells Prog cells hESCs hESCs
100 100 viability in% viability in % 50 50
0 0 0,01 0,1 1 10 100 1000 0,01 0,1 1 10 100 conc. in uM conc. in uM
Saccharin 5-Fluorouracil
IC50 hESC No IC50 at 1mg/ml IC50 hESC 0,044 µM
IC50 Prog No IC50 at 1mg/ml IC50 Prog 0,080 µM
IC50 hFF No IC50 at 1mg/ml IC50 hFF 0,304 µM Developmental toxicology - Retinoids
ATRA hFF 13CRA hFF 150 150 Prog cells Prog cells hESCs hESCs
100 100 viability in % 50 viability in % 50
0 0 0,01 0,1 1 10 100 0,01 0,1 1 10 100 conc. in uM conc. in uM
ATRA 13-cis retinoic acid
IC50 hESC 30,38 µM IC50 hESC 15,85 µM
IC50 Prog 0,001 µM IC50 Prog 0,0004 µM
IC50 hFF 133,7 µM IC50 hFF 301,6 µM Developmental toxicology - Cardiac differentiation
Contracting areas appear during spontaneous differentiation of hES cells and can be maintained for up to 3 months (~30-100bpm)
High variability in the human system – Not suitable as an endpoit Developmental toxicology - Improved human endpoints
• Recapitulate early human embryonic development in-vitro • Measure endpoints on protein level using novel mAbs • Measure endpoints on gene level using RT-qPCR or reporter cell lines (under development in collaboration with Invitrogen)
Marker 01 Marker 01 Ectoderm Marker 02 Marker 02
Marker 03 Marker 03 Differentiation Mesoderm Marker 04 Marker 04
Marker 05 Marker 05 Endoderm Marker 06 Marker 06
Undifferentiated In-vitro Development Germ Gene Protein hES cells layer level level Developmental toxicology - Novel monoclonal antibodies
• In-house derived mABs • Surface markers 10x 10x • Directed against undifferentiated hES cells and early lineage determination 20x 20x Novel monoclonal antibodies for hESC
10x 10x
20x 20x Developmental toxicity - Screening
Screening using monoclonal surface antibodies or engineered cell lines
Endoderm
Novel monoclonal antibodies for hESC Mesoderm
Ectoderm
t t t
Normal development Inhibition Directed differentiation Metabolism Metabolism - Hepatoblast/cyte-like cells from hES cells
Hepatoblast-like cells
Hepatocyte-like cells Metabolism - Hepatocyte-like cell characterization
A B ∗
∗ ∗ Glycogen storage C D C
α-1-AT AFP EF
Albumin CK18 G H
Foxa2 LFABP Metabolism - Induction experiments Phase 1 enzymes
) d ) M F e ( ( d t e d a d s s t e e e e s t e a c r c e t e t t y y r u n u c t y c d u d o n c t o n n o t u i 5 i a a . 2 n 2 2 2 i p 2 t p 0 0 0 G e 0 a e 0 0 0 p 0 r h h A A A A p He p S S S S Ke h h CYP1A
CYP3A
β-actin Metabolism - Induction experiments Phase 2 enzymes
s e m . r o t . d . ) s n r . ) o t F M r u in d ( n ( c 5 5 u n . . s i . . i 2 p p T m 2 2 7 7 e e 0 0 6 S r 6 h G F 0 0 1 1 h G e p E A A A A p p r v e li M S S S S h h h H
GST A1-1
GST M1-1
GST P1-1
b-actin
Söderdahl et al, submitted Thank you!