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).
Induced Pluripotent Stemcells: A P4 Perspective
Induced Pluripotent Stem Cells:<br />The Personal Stem Cell<br />Emile F. Nuwaysir, Ph.D.<br />Chief Operating Officer<br />Cellular Dynamics International<br />
Induced Pluripotent Stem Cells<br />Introduce four reprogramming factors<br />Blood draw or skin biopsy<br />Induced pluripotent stem (iPS) Cells<br />iPS cells are not the same as embryonic stem cells, and are NOT derived from fertilized embryos!<br />iPS cells are derived from adult tissue sample (skin or blood)<br />
Induced Pluripotent Stem CellsTwo Critical Characteristics<br />What’s so special about iPS cells?<br />Replicate Indefinitely<br />Make all 208 cell types in human body<br />Induced Pluripotent Stem (iPS) Cells<br />
Reprogramming: Science Magazine’s 2008 Breakthrough of the Year<br />
iPS Cells and Tissue Differentiation<br /><ul><li>Reprogamming factors are still present in genome of cell, making these iPS cells unsuitable as source for personal cell therapy</li></li></ul><li>iPS Cells and Tissue Differentiation<br /><ul><li>iPS 2.0 cells are superior because they are “footprint-free”… no residue remains after reprogramming process.</li></li></ul><li>iPS Cells and Tissue Differentiation<br />iPS cells are uniquely useful<br />Derived from adult tissue via non-invasive methods<br />Can be expanded indefinitely<br />Can be differentiated into any cell type in the body<br />iPS cells are superior to embryonic stem cells<br />Eliminates ethical issues regarding tissue source<br />Enables diversity of genotype and phenotype<br />Reprogram sample tissue into iPS cells<br />iPS cells multiply and expand in culture indefinitely<br />Draw 1 small sample from 1 person<br />Differentiate iPS cells into unlimited numbers any tissue cell in the body<br />
iPS Cells: Unlimited Potential<br />iPS cells<br />Represent any individual genotype<br />6 Billion People<br />208 Cell Types<br />Access to the human body<br />Access to Diversity<br /><ul><li> Disease phenotypes
Multiple samples because time-course may be informative
Source for your own cell therapy (autologous)</li></li></ul><li>3. DIFFERENTIATE <br /> Into heart (or never, liver, blood vessel) cells<br />Scalable iPS Manufacturing Process<br />2. EXPAND<br />IPS cell line --> 1 billion cells per day<br />1. CREATE<br /> iPS line from 1 person<br />4. PURIFY>95 purity<br />>10B heart cells / month<br />Scale-up to Enable First Stage of Research<br /><ul><li>Industrial pipeline to manufacture pure human tissue cells in necessary quantity, quality, and cost
Pipeline is applicable across all human tissue types
Utilizing ISO and GMP processes</li></li></ul><li>Parallel Manufacturing Process<br />PARALLEL PROCESSING <br /><ul><li>1000’s of individuals
Billions of tissue cells</li></ul>Scale-Out to Enable P4 Medicine<br /><ul><li>iPS cell lines for 1,000s of people
Population scale iPS cell banks (e.g. research cohort, country, etc.)
Tissue cells (nerve, liver, blood vessel cells, etc) from each individual’s iPS cell line</li></li></ul><li>iCell Cardiomyocytes<br /><ul><li>iCell™ Cardiomyocytes
Testimonials<br />“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...” <br />- Kyle Kolaja, global head of predictive toxicology screens and <br />emerging technologies for Roche. <br />Sep 23, 2010, BloomBergBusinessWeek, “Stem Cells Test <br />Drugs in a Dish, May Save Companies Millions”<br />Roche<br />"This is a game changer. It's going to dramatically change biology and drug development.” <br />- Sandra Engle, senior principal scientist at Pfizer. <br />Jan 22, 2010, MIT Technology Review<br />“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,” <br />- Jason Gardner, vice president and head of the stem cell drug performance unit at GlaxoSmithKline. <br />Sep 23, 2010, BloomBergBusinessWeek, <br />“Stem Cells Test Drugs in a Dish, May Save Companies Millions”<br />Pfizer<br />GlaxoSmithKline<br />
Express key molecular and functional attributes</li></ul>Typical morphology observed early after differentiation (left). Three weeks later, the neurons are more organized into extensive networks (below).<br />TH/Hoechst<br />Glutamate/Hoechst<br />GABA/Hoechst<br />
Disease Modeling with iCellCardiomyoyctes<br />An In Vitro Model for Hypertrophy<br />NPPB<br />mRNA <br /><ul><li>Under appropriate cell culture conditions, iCell cardiomyocytes can be induced into a state of hypertrophy that closely resembles cardiac hypertrophy in vivo (cell size, gene expression, etc).</li></li></ul><li>The Power of iPS Cells<br />A Case Study with Cardiomyocytes<br />Proposal to the National Heart, Lung, and Blood Institute in partnership with the HyperGen –ECHO Study<br /><ul><li>HypergenCohort - large, family-based, ECHO data for Caucasians and African-Americans.
Completed Genome-Wide Association Study - identified SNPs for Left Ventricular Hypertrophy (LVH) and other important phenotypes.
Novel approach- use iPSC-derived ventricular cardiomyocytes to study the molecular changes in the development of LVH.
First human cardiomyocyte-based cellular model of LVH.
Functional demonstration of LVH-associated SNPs will provide better disease modeling, management and treatment.</li></li></ul><li>The Power of iPS Cells<br />A Case Study with Cardiomyocytes<br />Investigative Approach<br /><ul><li>Produce iPSC lines from 250 selected cohort donors.
Produce 50M cryopreserved ventricular cardiomyocytes from each iPSC line.
Experimentally induce the LVH phenotype in culture.
Perform RNA expression analysis on genes and that are ‘transcriptional surrogates’ for LVH.
Determine the role of HyperGen SNPs in LVH epidemiology.
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.
The HyperGEN Cohort has a large variety of additional phenotypes; diabetes, hypertension, obesity, etc.</li></li></ul><li>Conclusions and Next steps<br /><ul><li>iPS Cells can be utilized to provide large numbers of highly pure cell models.
iPS technology will continue to evolve</li></ul>#’s of Lines will increase, better genetic models, diversity, etc.<br />New Parallel reprogramming method development<br />Reduce amount of somatic starting material<br />Understanding of impact of iPS cells derived from different somatic tissues<br />Next Steps:<br /><ul><li>Specific cell sub-types (every cell subtype in the human body)