validation of gene expression signatures employed in
TRANSCRIPT
Validation of Gene Expression Validation of Gene Expression Signatures Employed in Directed Signatures Employed in Directed Stem Cell DifferentiationStem Cell Differentiation with with Small Molecule PerturbagensSmall Molecule Perturbagens
Charisma N. GarciaCharisma N. GarciaCancer GenomicsCancer GenomicsSummer Undergraduate Research ProgramSummer Undergraduate Research Program
What is a stem cell?What is a stem cell?
Potential to differentiate into Potential to differentiate into numerous cell typesnumerous cell typesCapacity to selfCapacity to self--replicate indefinitelyreplicate indefinitelyWhen in culture, can remain in an When in culture, can remain in an undifferentiated state for many undifferentiated state for many generationsgenerations
EmbryonicEmbryonic stem cellsstem cells
Source: Inner cell Source: Inner cell mass of the premass of the pre--implantation implantation blastocystblastocyst–– Blastocyst:Blastocyst: 44--5 5
day embryoday embryo
NIH stem cell report, 2001
EmbryonicEmbryonic stem cellsstem cellsNIH Stem Cell Report, 2001NIH Stem Cell Report, 2001
PLURIPOTENT
Potential stem cell Potential stem cell applicationsapplications
Monitoring developmental biologyMonitoring developmental biologyGenetic engineeringGenetic engineeringPharmaceutical testingPharmaceutical testingToxicologyToxicologyTherapeutic transplantsTherapeutic transplants
–– Chronic heart diseaseChronic heart disease–– EndEnd--stage kidney disease stage kidney disease –– Liver failure Liver failure –– CancerCancer–– Parkinson’s diseaseParkinson’s disease–– Spinal cord injurySpinal cord injury–– Multiple sclerosisMultiple sclerosis–– Alzheimer’s diseaseAlzheimer’s disease–– Amyotrophic lateral sclerosisAmyotrophic lateral sclerosis–– DiabetesDiabetes–– Skin graftsSkin grafts–– Purkinje cell degenerationPurkinje cell degeneration–– Duchenne’sDuchenne’s muscular dystrophymuscular dystrophy–– OsteogenesisOsteogenesis imperfectaimperfecta
NIH Stem Cell Report, 2001NIH stem cell report, 2001
Directed differentiationDirected differentiation
Using laboratory Using laboratory techniques:techniques:
Unspecialized cellUnspecialized cell
Specialized cellSpecialized cell[determined by gene expression signatures, [determined by gene expression signatures,
i.e. expression of marker genes]i.e. expression of marker genes]
Small molecule perturbagen
http://www.stemcellresearchfoundation.org/index.htm
Microarray Gene Expression Profiling of Microarray Gene Expression Profiling of Embryonic Stem Cells and Dissected Embryonic Stem Cells and Dissected Embryonic Tissues for Marker SelectionEmbryonic Tissues for Marker Selection
Microarrays GE-HTS
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Nanog.ESUtf1.ESZfp42.ESTcfap2c.ESUpp1.ESPou5f1.ESEsrrb.ESEras.ESFbxo15.ESNr0b1.ESFoxa1.Emb_endoHas2.Emb_endoFoxg1.Emb_endoSix1.Emb_endoEmb.Emb_endoFoxa2.Emb_endoDlx3.Emb_endoIgfbp5.Emb_endoIrs4.Emb_endoPax9.Emb_endoSox9.Emb_endoKrt2-7.Emb_endoTcfap2a.Emb_endoCrb3.Emb_endoIsl1.Emb_endoTcfcp2l3.Emb_endoRab15.Emb_endoTmem46.Emb_endoNpnt.Emb_endoFrem2.Emb_endoCrabp2.Emb_ectoSox1.Emb_ectoNkx1-2.Emb_ectoEgr2.Emb_ectoHoxc4.Emb_ectoSfrp1.Emb_ectoTnfrsf19.Emb_ectoBoc.Emb_ectoSp8.Emb_ectoSox21.Emb_ectoMyocd.Emb_mesoActc1.Emb_mesoPdgfra.Emb_mesoEdnra.Emb_mesoFoxf1a.Emb_mesoMyl7.Emb_mesoAnkrd1.Emb_mesoFoxc2.Emb_mesoAsb4.Emb_mesoPrrx1.Emb_mesoHnf4a.EE_endoTtr.EE_endoIrf6.EE_endoCubn.EE_endoApob.EE_endoGata1.EE_ectoZfpn1a1.EE_ectoHmbs.EE_ectoEraf.EE_ectoTspan33.EE_ecto
EE Ecto
EE Endo
Emb Meso
Emb Ecto
Emb Endo
ES
ES D8 Emb Endo
D9 Emb Endo
D8 Emb Ecto
D8 Emb Meso
D8 EE Endo
Microarrays
Embryonic Embryonic Embryonic Embryonic Extra-embryonic Extra-embryonic HousekeepingStem cell Endoderm Ectoderm Mesoderm Endoderm Ectoderm Genes
Tcfap2c Foxg1 Egr2 Ankrd1 Hnf4a Tspan33 GapdhNr0b1 Tcfap2a Sox21 Foxc2 Ttr Hmbs ActbEsrrb Six1 Hoxc4 Prrx1 Irf6 Eraf Pgk1Fbxo15 Sox9 Nkx1-2 Asb4 Cubn Gata1 Ldh1Uppl Is11 Sox 1 Myl7 Apob Zfpn1a1Zfp42 Dlx3 Boc Actc1Pou5f1 Rab15 Sfrp1 EdnraNanog Frem2 Tnfrsf19 PdgfraUtf1 Has2 Sp8 MyocdEras Igfbp5 Crabp2 Fox1a
Tmem46Irs4Pax9NpntFoxa1Krt2-7EmbTcfcp2l3Foxa2
Gene Expression SignaturesGene Expression Signatures
Project OverviewProject Overview
1.1. Start and maintain a mouse Start and maintain a mouse embryonic stem cell (mES) line embryonic stem cell (mES) line
2.2. Treat the mES with the chosen Treat the mES with the chosen compounds, at various dilutions, compounds, at various dilutions, for 4 daysfor 4 days
3.3. Determine if differentiation Determine if differentiation occurred by comparing gene occurred by comparing gene expression signaturesexpression signatures
GOAL: Validate gene signatures in tissue culture
Treatments to be tested Treatments to be tested for differentiationfor differentiation
Treatment Hypothesized Target Cell Germ LayerAll-trans-Retinoic Acid Neurons Ectoderm5-Azacytidine Cardiomyocytes MesodermFluoxetine Hydrochloride Neurons EctodermMitomycin C Dopamine Neurons EctodermReversine Myocytes MesodermScriptaid Induces hemoglobin Mesoderm
DILUTIONS:6.4 µM3.2 µM1.6 µM0.8 µM0.4 µM0.2 µM0.1 µM0.05 µM
1 2 3 4 5 6 7 8 9 10 11 12
A DMSO LIF ATRA 0.05 µM 5AZA 0.05 µM * FLUOX 0.05 µM MITOC 0.05 µM * REVERS 0.05 µM SCRIP T 0.05 µM DMSO LIF
B DMSO LIF ATRA 0.1 µM 5AZA 0.1 µM * FLUOX 0.1µM MITOC 0.1 µM * REVERS 0.1 µM SCRIPT 0.1 µM DMSO LIF
C DMSO LIF ATRA 0.2 µM 5AZA 0.2 µM * FLUOX 0.2 µM MITOC 0.2 µM * REVERS 0.2 µM SCRIPT 0.2 µM DMSO LIF
D DMSO LIF ATRA 0.4 µM 5AZA 0.4 µM * FLUOX 0.4 µM MITOC 0.4 µM * REVERS 0.4 µM SCRIPT 0.4 µM DMSO LIF
E DMSO LIF ATRA 0.8 µM 5AZA 0.8 µM * FLUOX 0.8µM MITOC 0.8 µM * REVERS 0.8 µM SCRIPT 0.8 µM DMSO LIF
F DMSO LIF ATRA 1.6 µM 5AZA 1.6 µM * FLUOX 1.6 µM MITOC 1.6 µM * REVERS 1.6 µM SCRIPT 1.6 µM DMSO LIF
G DMSO LIF ATRA 3.2 µM 5AZA 3.2 µM * FLUOX 3.2 µM MITOC 3.2 µM * REVERS 3.2 µM SCRIPT 3.2 µM DMSO LIF
H DMSO LIF ATRA 6.4 µM 5AZA 6.4 µM * FLUOX 6.4 µM MITOC 6.4 µM * REVERS 6.4 µM SCRIPT 6.4 µM DMSO LIF
96 Well Layout96 Well Layout
Ligation-mediated amplification (LMA) & Gene expression-based highthroughput (GE-HTS) experimental overview
Treated cells
1231
ExpressionMarker
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TT
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TT
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TT
TTTT
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AAAAAA
AAAAAA
AAAAAA
AAAAAA
mR
NA
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AAAAAA
AAAAAA
AAAAAA
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TT
TTTT
TT
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TTRT
TTTT
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TTTT
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TTTT
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TTprobes
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ligation
PCRLuminex
primer site
primer site
complementaryto cDNA
complementaryto cDNA
probe specific tag
SAPE
Courtesy of Cristian Jitianu
Acknowledgements:Acknowledgements:
Todd Todd GolubGolubCristianCristian JitianuJitianu
Angela BrunacheAngela BrunacheBruce BirrenBruce Birren