HESI Workshop on Alternative Assays for Developmental Toxicology
Stem Cell Biology
Embryonic Stem Cells as Model System to Study Embryotoxic Effects
Cary, N.C. February 27- 28, 2007
Anna M. Wobus ([email protected]) Leibniz Institute (IPK) Gatersleben, Germany UniqueUnique PropertiesProperties ofof EmbryonicEmbryonic StemStem (ES)(ES) CellsCells
in vivo
Oocyte
Sperm Zygote
in vitro
Inner cell mass Cultivation (ICM) ES cells Proliferation
Blastocyst Cultivation Differentiation Primordial germ cells EG cells (PGC) "embryoid body" (EB)
Endoderm Ectoderm Liver cells Neuronal cells Pancreas Mesoderm cells Cardiac cells Skeletal muscle cells Blood cells Fat cells ESES cellscells differentiatedifferentiate inin vitrovitro intointo cellcell typestypes ofof allall threethree primaryprimary germgerm layerslayers
C D E
Mesoderm
Cardiomyocytes Skeletal muscle Smooth muscle A B F G H
Ectoderm
Neurons Glial Epithelial Embryonic stem cells Embryoid body I K L
Endoderm
Pancreatic cells Hepatocytes Hepatocytes ESES cellcell differentiationdifferentiation inin vitrovitro recapitulatesrecapitulates processesprocesses ofof earlyearly embryonicembryonic developmentdevelopment DifferentiationDifferentiation ofof ESES cellscells inin vitrovitro
1) Embryoid body formation (3 germ layers!) • ‘Mass culture‘ • ‘Hanging drop culture‘ • Methylcellulose • ‘Spinner culture‘
2) Differentiation in monolayer culture (neural cells)
3) Co-culture systems (PA-6, END-2) DifferentiationDifferentiation ofof ESES cellscells inin vitrovitro
1) Embryoid body formation (3 germ layers!) • ‘Mass culture‘ • ‘Hanging drop culture‘ • Methylcellulose • ‘Spinner culture‘
2) Differentiation in monolayer culture (neural cells)
3) Co-culture systems (PA-6, END-2) ParametersParameters thatthat affectaffect ESES CellCell DifferentiationDifferentiation
Culture media, supplements
Concentration and quality of FCS
ES cell line, passage number
Number of cells in the EBs
Time of EB plating
⇒ Threshold concentrations of ECM and growths factors in the EB regulate ES cell differentiation CCardiacardiac differentiationdifferentiation DifferentiationDifferentiation ofof beatingbeating cardiomyocytescardiomyocytes
Titin (Z-disk)-positive cardiac cells CardiacCardiac differentiationdifferentiation
ES cells EBs Multilineage Cardiac clusters progenitors
Stage 123 4 nestin/desmin titin
Culture (d) 055+65+24 Differentiation induction
Media Additives L-Glut, NEAA, MTG
Basal medium DMEM + 15%FCS IMDM + 20% FCS
Substrate MEF Gelatine or Laminin ExpressionExpression ofof mesodermmesoderm-- andand cardiaccardiac--specificspecific genesgenes duringduring ESES--derivedderived cardiogenesiscardiogenesis
1 2 3 4 5 6 7 8 9101214day T (Brachyury)
BMP-4
plating
ES 3 5 + 2 + 5 + 7 + 12 + 18 day
Nkx2.5
α−MHC
β−MHC
ANF
MLC-2V CardiacCardiac--specificspecific actionaction potentialspotentials andand ionion currentscurrents
Early Intermediate Terminal stage +60
0 mV Action
potentials -80 Atrial-like Ventricle- Sinusnodal- 1 sec like like
ICa + ++ ++ ++ ++
Ito + + + + +
IK,ATP + + + + +
IK - + + + +
INa - + ++ ++ +- I - - + + - Ion currents K1
IK,Ach - - + - + - If - + - ++ ESES--derivedderived cardiomyocytescardiomyocytes respondrespond pharmacologicallypharmacologically similarsimilar toto cardiaccardiac cellscells developeddeveloped inin vivovivo
Isradipin Nisoldipin Bay K Gallopamil CarbacholAtropin Forskolin Isoproterenol 8644 Diltiazem Dihydropyridine Ca2+ senstitive GTPγS GTPγS channel (L-type) Cholinoceptor Adenylyl cyclase β-adrenoceptor 2+ N N Ca
GGi s Gs Phoshat- P ases C N ATP CCcAMP N cAMP-dependent PDEs protein kinase A C ATP Ca2+
PDEs inhibitors 8-bromo PKA PKA ATPγS okadaic IBMX, EHNA, cAMP inhibitor catalytic acid milrinone, rolipram peptide subunit ESES--derivedderived cardiomyocytescardiomyocytes forfor drugdrug screeningscreening
Embryoid body (EB) Beating clusters in EB outgrowths
Estimation of basal level of beating frequency by the LUCIA 'Heart' Pharmacological analysis imaging system of cardioactive drugs
BayK 8644 Diltiazem
100 Diltiazem BayK 8644 Dose-response-effects 80 60 40 20 0 -20
Schlagfrequenz (Schläge/min) -40 -60
-8 -7 -6 -5 log Koncentration (M) NNeuronaleuronal differentiationdifferentiation NeuronalNeuronal differentiationdifferentiation
ES cells EBs Neural Neuronal Dopaminergic progenitors cells neurons
Stage 12 3 4 5 nestin ß III-tubulin TH
Culture (d) 0 4 4+7 4+14 C 4+30 Selection Expansion Differentiation induction
Media Additives B1 (-FCS: 4+1) B2 + EGF;bFGF Neurobasal + B27 (2%) + SPFs
Basal medium IMDM+20%FCS DMEM/F12 DMEM/F12 +10%FCS
Substrate Gelatine poly-L-Ornithine/Laminin 100 ES EG C 80
60 cells (%)
ESES cellscells differentiatedifferentiate 40
20 nestin-positive
intointo neuralneural precursorprecursor Percentage cell-derived of ES 0 4+ (ES) 2681012144 cellscells andand TH+TH+ (EG)5+ dopaminedopamine--producingproducing EB plating Replating 100 -SPF E neuronsneurons 80 + SPF
60
Immunofluorescence analysis nef ostin-positive neuronaprl ecurso cells (A,B,C), tyrosinhye droxyla(Tse H)- (D,E,) andopad mine transport(Der AT)-positivdoe paminergic neurons (F,G) * * 40 labelled cells
TH+- (%)Syn- of cells 20
0 4+ 16 23 30
100 -SPF + SPF G 80
60 * 40 * labelled cells
DAT+-Syn- (%) of cells 20
0
4+ 16 23 30 Time of differentiation (d) Increase of Nurr1, TH and bcl-2 mRNA levels by SPFs
Embryoid bodies plated at day 4 Genes 4+ 4 7 12 16 16 23 23 30 30 35 35 40 40 44 44 En-1 A
D2R-L TH Nurr1 bcl-2 β-tubulin
100 80 En-1 B 60 40 20 0 100 80 D2R-L C 60 40 20 R-L, TH, Nurr1 and
2 0 100 TH D 80 * 60 40 20 0 100 Nurr1 80 * ** ** E 60 40 20 0 100 bcl-2 F 80 ** * 60 ** 40 20 Relative mRNA levels of En-1, D bcl-2 (%) 0 4+ 4 7 12 16 23 30 35 40 44 Time of Differentiation -SPF (4+14-4+44d) +SPF (4+14-4+44d)
------5 . 7 3
D ( o p a m i n e )
Retention time (min) Cultures with SPF with Cultures
------5 . 6 9
D ( o p a m i n e thout SPF thout ) Retention time (min) Cultures wi
12345 678 12345 678
---- 5 . 7 9
(D o p a m i n e analyzed by HPLC ) analyzed by HPLC Retention time (min) External standard Dopamine synthesis of TH+ neurons Dopamine synthesis of TH+ neurons 012345 678 0
600
I n te n s ty i
( m v 1200 ) NeurotoxicNeurotoxic effectseffects ofof 100100 μμMM MPTPMPTP onon TH+TH+ neuronsneurons
A B
C D Control MPTP treatment 48h PancreaticPancreatic differentiationdifferentiation PancreaticPancreatic DifferentiationDifferentiation ofof wtwt andand Pax4+Pax4+ ESES CellsCells
ES cells EBs Multilineage Committed Islet-like progenitors progenitors clusters
Stage 12345
Nestin/CK19 C-peptide/Nestin Insulin/C-peptide
Culture (d) 0 5 5+9 5+16 5+28 Differentiation induction
Media Additives L-Glut, NEAA, MTG NA + Laminin
Basal medium DMEM + 15%FCS IMDM + 20% FCS N2 + B27 (- FCS: 5 + 10d to 5 + 28d)
Substrate MEF Gelatine poly-Ornithine/Laminin UpregulationUpregulation ofof pancreaspancreas--specificspecific genesgenes andand differentiationdifferentiation ofof isletislet--likelike clustersclusters
ES 5+9d 5+16d 5+28d panc brain Insulin/ proinsuli n InsulinInsulin II I Glucagon Amylase Somatostatin ß- tubulin C-peptide/insulin Pdx1 Pax4 IAPP ß-tubulin 5+95+16 5+28 5+28d wt Time of differentiation (d) ESES cellscells (Pax4+)(Pax4+) differentiatedifferentiate intointo functionalfunctional insulininsulin--producingproducing cellscells
Insulin/C-peptide Voltage-activated Na+ channels ATP-sensitive K+ channels (Pax4+ cells)
Wt 5+28d 30 Transplantation
Insulin/C-peptide 25
20
15
10
Blood glucose [mM] wt (n=6) Sham control (n=6) + 5 Voltage-activated K channels + + STZ Pax4 (n=5) Pax4 , graft removal (n=2) 0 + 0481216202428323640 Pax4 5+28d Time (days) Bar = 20 µm
Blyszczuk et al., PNAS 2003; 2004; Kania et al., IJDB 2004; Schroeder et al., Nature Prot. 2006 ESES cellscells recapitulaterecapitulate thethe programprogram ofof mesodermalmesodermal,, ectodermalectodermal andand endodermalendodermal lineagelineage developmentdevelopment
Undifferentiated ESC Inner cell mass of blastocyst
Proliferation and differentiation
Cardiac cells Neuronal cells Pancreatic cells
Wobus et al. (1991) Rolletschek et al. (2001) Blyszczuk et al. (2003) Wobus & Boheler, 2005 etc…. Blyszczuk et al. (2004) etc…. ESES CellCell TechnologyTechnology inin vitrovitro
Embryotoxicity/Teratogenicity Cell Therapy Selective Differentiation Blastocyst Analysis of Embryotoxic Factors in vitro and Isolation
'G a in -o f- Injection Cultivation func In vitro t ++ io ES cells (A /A ) n Endoderm '
Ectoderm Pharmacology Differentiation Drug Screening embryoid body Mesoderm
n' io Homologous ct fun Recombination f- -o A- oss 'L
A-/A- Genomics/ Proteomics
- cDNA Arrays/Microchips - SAGE CriticalCritical parametersparameters ofof embryotoxicityembryotoxicity teststests usingusing ESES cellscells
• Treatment time (development-specific effects) • Concentration-dependent effects (threshold concentration) • Activity of chemicals under in vitro conditions (metabolic activity) • Genetic and epigenetic factors (long-term cultivation) • Several endpoints and lineages should be included into the test system. EffectsEffects ofof RARA onon ESES CellCell DDifferentiationifferentiation
• Biologically active form of Vitamin A • Highly teratogenic in animals and human • Malformations of craniofacial structures and CNS • Cardiac (conotruncal heart, aortic arch, ventricular septum) defects (RAR-/- and RXR-/- mice!) StageStage-- andand concentrationconcentration--dependentdependent effectseffects ofof RARA onon ESES cellcell differentiationdifferentiation RARA (10(10–9M,M, 55--7d)7d) inducedinduced upregulationupregulation ofof αα--cardiaccardiac MHCMHC andand MLCMLC--2v2v mRNAmRNA levelslevels
Control 10-9 M RA 10-8 M RA RARA (10(10-8/-9 M,M, 55--7d)7d) increasedincreased thethe numbernumber ofof cardiaccardiac ventricleventricle--,, butbut decreaseddecreased pacemakerpacemaker--likelike cellscells
control RA treatment ESES CellCell TestTest forfor CardiacCardiac--specificspecific EmbryotoxicityEmbryotoxicity
RSV-LTR Neor pGNA/MLC-2.1 Kpn I Reporter β-gal 2.1 MLC-2v Promoter Xmn I Transfection into ES cells
Differentiation (+ test substance: RA)
Embryoid body
Plating Differentiation (+ test substance: RA) a - RA (control) b + RA
Wobus et al., (1994), (1997); rev. Wobus & Boheler, Phys. Rev. (2005) EffectsEffects ofof LithiumLithium onon ESES CellCell DifferentiationDifferentiation
• Highly teratogenic in animals and humans • Induction of dorsoventral patterning in Xenopus • Treatment of manic depressive psychosis, psychiatric disorders • Lithium is able to cross the placenta LithiumLithium (5(5--10d)10d) decreaseddecreased cardiaccardiac andand skeletalskeletal musclemuscle cellcell differentiationdifferentiation ofof ESES cellscells αα--MHCMHC andand myoDmyoD mRNAmRNA levelslevels areare downdown regulatedregulated atat highhigh concentrationsconcentrations ofof LithiumLithium NeuralNeural--specificspecific SynSyn andand NFMNFM mRNAmRNA levelslevels areare partiallypartially upup regulatedregulated byby LithiumLithium
Schmidt et al. Differentiation (2000) SummarySummary ofof RARA andand LithiumLithium effectseffects onon ESES cellcell differentiationdifferentiation
• RA and Lithium show concentration- and stage- dependent effects on lineage differentiation of ES cells;
• RA and Lithium could induce the differentiation of specific cellular subtypes, but inhibit other lineages;
• ES cells are suitable cellular models to analyze embryotoxic effects of chemicals, if applied under defined conditions. EffectsEffects ofof PhysicalPhysical FactorsFactors:: ElectromagneticElectromagnetic FieldsFields (EMF)(EMF) onon ESES CellCell DDifferentiationifferentiation 1.1. EffectsEffects ofof highhigh frequencyfrequency EMFEMF (GSM,(GSM, 217217 Hz,Hz, 1.711.71 GHz)GHz) onon wtwt andand p53p53--//-- ESES cellscells
Analysis of the influence of the genetic background on EMF response of cells, effects of different modulation schemes
2.2. EffectsEffects ofof lowlow frequencyfrequency EMFEMF (50(50 Hz)Hz) onon wtwt andand p53p53--//-- ESES cellscells Analysis of 50 Hz power-line EMF: at different flux densities (0.1, 1.0, 2.3 mT), exposure times, and intermittency schemes EMFEMF exposureexposure toto undifferentiatedundifferentiated andand differentiatingdifferentiating wtwt andand p53p53 --//-- ESES cellscells
Undifferentiated ES cells (D3) cultivated on feeder layer
AB Cultivation of ES cells in hanging drops Cultivation of ES cells 0-2d in hanging drops for early differentiation 6h/48h EMF exposure Formation of embryoid bodies (EBs) 6h EMF exposure
RT -PCR analysis Cultivation of EBs in suspension 2-5d Upregulation of stress-
Plating of EBs 5d response, cell cycle and apoptosis regulating genes in p53-/- cells: 5 to 5+15d Differentiation hsp 70!, p21, c-jun, c- myc/ egr-1, bcl-2, c-jun
RT -PCR analysis Czyz et al., Bioelectromagnetics (2004); Mutation Res. (2004) 3.3. EffectsEffects ofof EMFEMF onon ESES--derivedderived neuralneural progenitorprogenitor cellscells
Influence of RF- and ELF- EMF on transcript levels and cellular functions of wild type ES-derived neural cells: Q-RT-PCR, IF, proliferation, differentiation, cell cycle, subG1 fraction, apoptosis, cytogenetic effects, COMET assay ES cells cultivated on feeder layer AnalysisAnalysis ofof EMFEMF exposureexposure Days Cultivation of ES cells in hanging drops 0d onon ESES--derivedderived neuralneural A Formation of embryoid bodies (EBs) 2d progenitorprogenitor cellscells
Transfer of EBs to bacteriological 2d-4d plates and cultivation in suspension
Plating of EBs (A) onto gelatin-coated plates 4d
Cultivation in ITSFn medium 4+1d
4+4d Q-RT-PCR EMF exposure 6h / 48h COMET, CA, SCE, M1:M2:M3 ratio, 4+6d subG1 FACS, IF (nestin+ / BrdU+ cells), mitochondrial function
B nestin Dissociation of nestin+ cells (B), 4+7d COMET, Q-RT-PCR, subG1 FACS replating onto poly-L-ornithine/ laminin-coated plates in 'nestin+ 4+8d COMET, subG1 FACS cells expansion medium'
4+11d Q-RT-PCR, subG1 FACS
Cultivation in Neurobasal medium with 10% FCS, 2% B27-supplement and 4+14d survival promoting factors C ßIII-tubulin D GFAP 4+17d Q-RT-PCR
4+23d Q-RT-PCR, subG1 FACS, IF Differentiation into neurons (C) and glia (D) (ßIII tubulin, TH, GFAP) Pluripotent ES cells
Formation of embryoid bodies (EBs)
Differentiation into neural progenitor cells SummarySummary
ELF-EMF RF-EMF RF- and ELF-EMF (6h) (50 Hz Powerline, 2.0 mT) (217 Hz, 1.71 GHz, 1.5 W/kg) affected transcript levels of Q-RT-PCR, COMET, CA, SCE, M1:M2:M3 ratio, subG1, IF (BrdU+ / regulatory genes, nestin+ cells), mitochondrial function (Mitotracker CM-H2X ROS) but not proliferation and
Differentiation into neuronal and glial cells differentiation of neural cells
Q-RT-PCR, subG1 FACS, IF (ßIII-tubulin, TH, GFAP)
ELF-EMF: RF-EMF: • Up-regulation of bcl-2 and bax • Up-regulation of bax, GADD45 • Down-regulation of GADD45 • Down-regulation of Nurr1 • No effects on DNA breakage, CA, • No effects on CA, SCE, subG1, SCE, subG1, mitochondrial function mitochondrial function • Primary DNA damage in Comet assay? Nikolova et al., FASEB J. (2005) EmbryotoxicityEmbryotoxicity TestsTests UsingUsing ESES CellsCells
• Conclusion: – Test substances should be applied during spontaneous differentiation of ES cells in vitro – Use of genetically modified ES cells (“Reporter strains”) – “Loss-of-function” or “gain-of-function” studies – Establishment of screening systems and high throughput applications (imaging analysis, FACS) • Critical Parameters: – Treatment time (stage-specific effects) influence lineage differentiation of ES cells – Concentration effects are dependent on treatment time or developmental stage of ES cells. AcknowledgementsAcknowledgements ‘In Vitro Differentiation‘ Group, IPK Gatersleben,Germany
Collaborations:Collaborations: KennethKenneth Boheler Boheler (NIA,Baltimore), (NIA,Baltimore), MarjanMarjan Rupnik Rupnik (Göttingen)(Göttingen) UrsulaUrsula Ravens,Ravens, IhorIhor Zahanich Zahanich (Dresden) (Dresden) NielsNiels Kuster Kuster (ETH (ETH Zurich)Zurich) Funding:Funding: VERUMVERUM Foundation,Foundation, DFG,DFG, BMBF,BMBF, EU:EU: REFLEXREFLEX