1. Induced Pluripotent Stem Cells: The Personal Stem Cell Emile F. Nuwaysir, Ph.D. Chief Operating Officer Cellular Dynamics International
2. Induced Pluripotent Stem Cells Introduce four reprogramming factors Blood draw or skin biopsy Induced pluripotent stem (iPS) Cells iPS cells are not the same as embryonic stem cells, and are NOT derived from fertilized embryos! iPS cells are derived from adult tissue sample (skin or blood)
3. Induced Pluripotent Stem CellsTwo Critical Characteristics What’s so special about iPS cells? Replicate Indefinitely Make all 208 cell types in human body Induced Pluripotent Stem (iPS) Cells
50. Express all function cardiac markers, including critical ion channels
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52. iCell Cardiomyocytes In Vitro Human Electrophysiology Cells Display Normal Ionic Currents Cells Display Normal Action Potentials Action Potential Examples Atrial-like Nodal-like Ventricular-like Control Tracings 30nM E4031 Control Drug Responses 3 nM Nifedipine 10 nM Nifedipine Control 100 ms
54. Testimonials “Had stem cell-derived heart tissue been available… the company could have pulled the plug early, saving two years of work and millions of dollars...” - Kyle Kolaja, global head of predictive toxicology screens and emerging technologies for Roche. Sep 23, 2010, BloomBergBusinessWeek, “Stem Cells Test Drugs in a Dish, May Save Companies Millions” Roche "This is a game changer. It's going to dramatically change biology and drug development.” - Sandra Engle, senior principal scientist at Pfizer. Jan 22, 2010, MIT Technology Review “Human heart cells that beat in a dish have never before been available in large quantity and consistent quality. Because they are functional human cells, they may provide a more accurate way of testing drugs than using rodents or the preserved human cells that have been available for lab research in the past,” - Jason Gardner, vice president and head of the stem cell drug performance unit at GlaxoSmithKline. Sep 23, 2010, BloomBergBusinessWeek, “Stem Cells Test Drugs in a Dish, May Save Companies Millions” Pfizer GlaxoSmithKline
59. Express key molecular and functional attributesTypical morphology observed early after differentiation (left). Three weeks later, the neurons are more organized into extensive networks (below). TH/Hoechst Glutamate/Hoechst GABA/Hoechst
71. iCell Hepatocyte CharacterizationFunction Lipid Storage Glycogen Storage Albumin Production 3x induction PAS Oil Red 2x induction 60% of iCell Hepatocytes store lipid (BODIPY assay) CYP3A4 Protein Expression CYP3A4 Activity
82. Provides a promising approach to test GWA results and common DNA variation in the context of a cell-based system by directly assaying molecular function and effects.
Response to cytokine stimulation causesupregulation of adhesion molecules
Points to emphasize: Slide shows a summary of key hepatocyte characteristics that we are looking for in our iPS cell-derived hepatocytesThis image is of CDI’s iCellHepatocytes
Albumin Production Points to emphasize: iPS cell-derived hepatocytes expression levels of albumin protein that are similar to PHH, and much greater than HepG2 cells (top left panel)Albumin was measured via ELISAOther things to know: Albumin is measured and expressed as pg/ALB+ cell/day Number of ALB+ cells was determined from the actual wells that were assayed after 24-48 h of incubation, not from the number of hepatocytes seeded. Glycogen & Lipid Storage Points to emphasize:iPS cell-derived hepatocytes store glycogen, as measured by Period Acid Schiff (PAS) staining (top middle panel)iPS cell-derived hepatocytes store lipid, as measured by Oil Red staining (top right panel) Lipid storage has also been measured using a BODIPY assay, and 60% of the iPS cell-derived hepatocytes store lipid (data not shown)Other things to know: Cells are counter-stained with hemotoxylin (purple nuclei)CYP3A4Points to emphasize: Data show CYP3A4 protein expression and activity Bottom left panel: see characteristic cytoplasmic staining of CYP3A4 (red) vs. nuclear staining with DAPI (blue). Bottom right panel: CYP3A4 activity measured in iCellHepatocytes and HepG2 cells using Promega’s P450-Glo assay (IPA substrate which is 1000x more specific to CYP3A4 vs. CYP3A5 or CYP3A7). This assay (and the P450-Glo assay for CYP1A2) is used routinely at CDI for monitoring P450 activity during development. Once highly pure cells are generated, P450 activity (basal and induced) will be measured by mass spec through external collaborations Cells were induced with 10uM DEX. Other things to know: Data represent the average of 3 lots of iPS cell-derived hepatocytes and 6 lots of HepG2 cells (various passages)iPS cell-derived hepatocytes express CYP3A7 mRNA, but no protein is detected by immunoblot and no activity is detected using Promega’s P450-Glo assay specific for CYP3A7).
