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Induced pluripotent stem cells as models of human genetic diversity and disease

Induced pluripotent stem cells as models of human genetic diversity and disease. Ronald P. Hart Rutgers Stem Cell Research Center Department of Cell Biology & Neuroscience NIMH Stem Cell Center November 3, 2011. Induced Pluripotency: From Gurdon to Yamanaka.

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Induced pluripotent stem cells as models of human genetic diversity and disease

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  1. Induced pluripotent stem cellsas models of human genetic diversity and disease Ronald P. Hart Rutgers Stem Cell Research Center Department of Cell Biology & Neuroscience NIMH Stem Cell Center November 3, 2011

  2. Induced Pluripotency: From Gurdon to Yamanaka In 1958 John Gurdon demonstrated nuclear transplant of somatic cell, leading to development of adult frogs Jaenisch, Daley, Studer, Eggan all worked on adapting concepts of nuclear transplant to cell fusion to create stem cells, inspiring… Yamanaka to screen transcription factors as the underlying mechanism of reprogramming, announcing the creation of mouse iPSC in 2006 With both Yamanaka and Thomson reporting human iPSC in 2007

  3. What are iPSC? • Adult somatic cells reprogrammed to become pluripotent • Exogenous expression of: • c-Myc, Sox2, Oct4, Klf4 (Yamanaka) • Oct4, Sox2, Nanog, Lin28 (Thomson) • c-Myc  l-Myc (less oncogenic) • + Glis1 (maternal mRNA; enhanced efficiency) • Delivered by: • Retrovirus • Lentivirus • Episome • Sendai • Small Molecules

  4. iPSC as disease models Wichterle & Przedborski (2010) Nat Neuro 13:800 iPSC from affected patient cells Genotype causing disease phenotype Differentiate to affected tissue Mechanism? Screen therapies? (ESC would require acquiring disease genotype from affected family without knowing symptoms/penetrance)

  5. Examples of disease modeling Inoue & Yamanaka (2011) Clin Pharm & Ther 89:655

  6. Relationship with other models Dolmetsch & Geschwind (2011) Cell 145:831 Better human relevance than animal models Similar mechanistic insight Higher throughput than patient studies

  7. Genetic diversity HOWEVER, small numbers of iPSC lines do not recapitulate human genetic diversity! Need to scale up production of iPSC Draw from genetically diverse sources

  8. NIMH Stem Cell Resource at RUCDR • Selected projects: • NIDDK • Diabetes Type I and Type II (also HBDI) • Inflammatory Bowel Disease • Kidney and Liver Diseases • NIMH • Alzheimer Disease • Autism (also CAN/AGRE) • Bipolar Disorder • Schizophrenia • Pharmacogenetic (clinical) trials • NIDA • Tobacco • Opiates • Cocaine • Clinical trials • NIAAA / COGA • Alcoholism • Simons Simplex Collection • Autism • Immune Tolerance Network • RUCDR is the world’s largest genetic repository, holding 4.5M specimens • >100 Technical Staff • 35,000 sq. ft. laboratory and storage space • Distribute ~ 1M samples for discovery based research • Automated workflow (annually): • >200,000 Nucleic Acid Extractions • >5M QPCR reactions • >10,000 DNA Microarrays • All samples linked with diagnostic data (nimhgenetics.org @ Wash. U.) • Many samples have been used in GWAS • NIMH Stem Cell Resource • Began operations June 2011 • Receive, expand, and bank source cells for iPSC formation • Prepare iPSC, validate, bank, and distribute • Advanced quality control: • RUID™ 96-SNP every sample • Routine myco & pathogen testing • High viability freezing protocol • STARLims tracking & process control • Custom qPCR panel for pluripotency, differentiation (neural lineage-enriched) • Karyotype & aCGH for CNV • FACS surface markers • Teratoma formation • Fee-for-service available • nimhstemcells.org

  9. iPSC produced at Rutgers • >14 iPSC lines • Retrovirus/Fibroblasts • Schizophrenia • Autism • Sendai/Lymphocytes • Planned/in progress • Addiction genes (NIDA) • Alzheimer’s (in review) • Tuberous sclerosis

  10. Neuronal cultures from iPSC VGLUT1/MAP2/Hoechst Na & K channels Voltage-clamped Immunocytochemistry Action potentials Current-clamped Lentiviral tagged SYN-GFP GFAP-GFP Hernandez, D’Ecclessis, Paradiso, Firestein

  11. Hypothesis testing: Gene upregulated in Schizophrenia should inhibit neuronal processes GFP Overexpress “Gene X” Hernandez, Firestein

  12. Neurotox: Dose-Dependent Response IC50=0.14μM IC50=2.2μM Ramirez, Richardson

  13. Summary • iPSC technology available as a contract service • RUCDR has large numbers of specimens with documented diagnoses, many GWAS-ed • Focus on neurogenesis • Neural-restricted precursors & mixed neuronal cultures • Imaging/cytochemistry/morphology • Lentiviral vectors carrying promoter-GFP for simple, high-throughput fluorescence assays of neurogenesis • Electrophysiology • Scaled to 96-well standard live/dead assays for neurotoxicology

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