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Induced Pluripotent Stemcells: A P4 Perspective
- 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
- 4. Reprogramming: Science Magazine’s 2008 Breakthrough of the Year
- 8. Hypo- or hyper-responders
- 9. “In vitro clinical trials”
- 10. Any genotype as background for molecular research and medicine… a first for humanity
- 11. Cardiomyocytes
- 12. Hepatocytes
- 13. Neurons
- 15. Any cell type for research and medicine… a first for humanityDifferentiate into all 208 cell types in the human body
- 19. Panels (in vitro Clinical Trials™)
- 20. Ethnic diversity
- 22. Disease models
- 23. Genetic
- 24. Engineered
- 25. Induced
- 26. Retrospective analysis of clinical trials
- 28. Autologous cell TherapySource: CDI Analysis
- 30. Individual drug toxicology (safety)
- 31. Individual drug pharmacology (efficacy)
- 32. Disease incidence or progression in target tissue
- 33. Preventive
- 34. Adverse drug reactions or disease
- 35. Personalized
- 36. 100% personal to you…
- 37. Participatory
- 39. Multiple samples because time-course may be informative
- 41. Pipeline is applicable across all human tissue types
- 44. Population scale iPS cell banks (e.g. research cohort, country, etc.)
- 46. Human cardiomyocytes from iPS cells
- 47. 99% pure, cryopreserved, ready to use.
- 48. Complete solution; cells, media, protocols
- 49. Available in virtually unlimited quantities
- 50. Express all function cardiac markers, including critical ion channels
- 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
- 53. iCell CardiomyocyteSingle Cell Pharmacology AP train Selected APs EADs
- 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
- 56. Human neurons from iPS cells
- 57. >95% pure, cryopreserved, ready to use.
- 58. Complete solution; cells, media, protocols
- 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
- 61. Human endothelial cells from iPS cells
- 62. >95% pure, cryopreserved, ready to use.
- 63. Full product solution; cells, media, protocols
- 64. Express key molecular and functional attributesZO-1-488 (green); Hoechst (blue) Inflammation Angiogenesis Stimulated Unstimulated
- 66. Human hepatocytes cells from iPS cells
- 67. >95% pure, cryopreserved, ready to use.
- 68. Full product solution; cells, media, protocols
- 69. Express key molecular and functional attributes
- 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
- 73. Completed Genome-Wide Association Study - identified SNPs for Left Ventricular Hypertrophy (LVH) and other important phenotypes.
- 74. Novel approach- use iPSC-derived ventricular cardiomyocytes to study the molecular changes in the development of LVH.
- 75. First human cardiomyocyte-based cellular model of LVH.
- 77. Produce 50M cryopreserved ventricular cardiomyocytes from each iPSC line.
- 78. Experimentally induce the LVH phenotype in culture.
- 79. Perform RNA expression analysis on genes and that are ‘transcriptional surrogates’ for LVH.
- 81. Determine the role of HyperGen SNPs in LVH epidemiology.
- 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.
- 85. Maturity (make terminal cells more mature)
- 86. Disease phenotypes (derive more disease models)
- 87. Organotypic biology (2D vs. 3D)
- 88. Mutlicellular systems (Systems Biology) to “reconstitute” the organ
Notas del editor
- Depolarizing currents, Na+ and Ca++
- 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).