Induced Pluripotent Stem Cells: an Introduction Oren Caspi MD, PhD
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1 Induced Pluripotent : an Introduction Oren Caspi MD, PhD The Sohnis laboratory for cardiac electrophysiology and regenerative medicine The Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology and internal medicine B, Rambam Health care campus
2 Presenter Disclosure Information Nothing to disclose
3 Induced Pluripotent stem (ips) cells Myocardial Regeneration Drug Screening & Development Disease Modeling
4 Human embryonic stem cells Potential Applications
5 Human embryonic stem cell derived cardiomyocytes (hesc-cm) Trophoectoderm Inner Cell Mass (ICM) ES cells colonies ANF Potential Applications Embryoid bodies in suspension Contracting areas Kehat et al. J Clin Invest Aug;108(3):
6 Caffeine puff Cardiac Excitability & Calcium handling Satin J.., Gepstein L. J Physiol Tissue engineering Caspi. O, Gepstein, Levenberg. Circ Res Potential Applications Directed Differentiation and Progenitor Cell Isolation Yang et al. Nature D model for drug screening Caspi O.., Gepstein L. Dev. 2008
7 Proof of Concept Cardiac Regenerative Tx. * Host-Graft coupling with the injured myocardium Shiba Y. Laflamme M., Nature 2012 Potential Applications Cell transplantation to the infracted heart Caspi O., Gepstein L. J Am Coll Cardiol Biological pacemaking Kehat I., Gepstein L. Nature Biotech, 2004
8 Limitations of The hesc model Generation of similar cell lines from adult individuals. Regenerative medicine: Non autologous cells= Immune Rejection Disease Modeling: Patient/Disease Specific Cell lines Potential Applications
9 Induced Pluripotent Stem (ips) Cells Potential Applications
10 Human Induced Pluripotent Stem (ips) Cells Jaenisch et al. Cell 2008 Potential Applications Nelson, TJ Nat. Rev. Cardiol. 2010
11 Cons Pros hips Cells derivation methodologies C-Myc OCT-4 SOX-2 KLF-4 Lentiviral or Retroviral vectors Excisable transgenes: Viral: Cre-LoxP Non-Viral: PiggyBac Non- Integrating Viral vectors Episomal Plasmids & Minicircle vectors Polyargenine peptide fused proteins Reproducible Transduces Non dividing cells Most common Reduced risk for transgene reactivation. Sendai- DNA free, relatively efficient even in primary cells. Lower risk for integration RNA/DNA free Genomic integration Incomplete silencing Low efficacy of excision Sendai-Purging cells with replicating viruses Occasional integration may occur Low efficiency Efficiency Clinical Safety 1:10 2 1:10 7
12 hips Cells derivation methodologies Improving efficiency and safety shrnas, BIX01294, Vit. C Somatic Cells Histone methylation GSK VPA Histone deactivation DNA Methylation TGF-b 5-AZA,RG- 108 microrna Modified RNA MiR-200c Oct-4, SOX2 MiR-302s KLF4, c-myc Reprogramming mir-369s or 367 ips Cells ERK Potential Applications Warren et al. Cell stem Cell 2009 WNT3a, CHiR AZA,RG- 108 PD
13 Cardiac differentiation of hipsc Fibroblasts hips Cells Caffeine puff ctni 1 F/F 0 α-actinin Potential Applications Itzhaki & Rapoport et al. Plos ONE sec Zwi & Caspi et al., Circulation 2009
14 Directed Cardiac Differentiation Kattman et al.. Cell Stem cells 2011 Dubois et al. Nature Biotech 2011 Potential Applications
15 Clinical Applications of h Potential Applications Robinton et al. Nature 2012
16 Myocardial Regeneration with hips Cells ctnt HF-hiPSC derived cardiomyocytes Matsuura et al. Biochem Biophys Res Commun Neonatal rat cardiomyocytes cx43 Myocardial Regeneration Streckfuss-Bomeke et al. Eur Heart J Zwi-Dantsis, et al. Eur Heart J 2012
17 Drug Screening & Discovery Reprogramming Somatic Cells ips Cells Drug Screening & Discovery Safer Tx. Personalized Tx. Novel disease Specific Tx. Safety & toxicity Screening ( QT, Cardiotoxic Tx.) Individualizing Drug Tx. Target identification By Disease-in-dish Assays Patient/Disease Specific
18 ipsc based Disease Modeling ARVC Cardiomyopathy DCM, Leopard LQTS (1,2,8) CPVT 1 CPVT2 Disease Modeling Pompe
19 30 mv APD (msec) APD (msec) APD (msec) Disease Modeling- Long QT * Ventricular * * 500 Ventricular Atrial Nodal 0 APD 50 APD 70 APD 90 Con Control 0 mv * Atrial * * LQTS 0 mv APD 50 APD 70 APD 90 Nodal Con Disease Modeling 400 ms APD 50 APD 70 APD 90 Cont Itzhaki I, Maizles L, Huber I et al. Nature 2011
20 20 mv Disease Modeling- Long QT 2 Development of Triggered-Activity in the LQTS hipscs-cms EAD EAD Triggered beat Triggered beat 5 sec Disease Modeling Itzhaki I, Maizles L, Huber I et al. Nature 2011
21 30 mv 20 mv Disease Modeling- Long QT 2 Pinacidil (K ATP Channel Opener) Ameliorating Effects on the LQTS-hiPSCs-CMs LQTS Baseline LQTS Pinacidil LQTS Baseline LQTS Pinacidil Disease Modeling 1 sec 5 sec Itzhaki I, Maizles L, Huber I et al. Nature 2011
22 Disease Modeling Arrhythmogenic Right Ventricular Cardiomyopathy ARVC Disease Modeling Basso et al. Lancet 2009
23 Disease Modeling Arrhythmogenic Right Ventricular Cardiomyopathy nm nm D ARVC S M D D D S Desmosomal gap width Control * ARVC Total desmosome width ** Disease Modeling 200nm Control 200nm ARVC Control ARVC
24 Disease Modeling Arrhythmogenic Right Ventricular Cardiomyopathy ARVC p<0.05 Disease Modeling
25 Disease Modeling Arrhythmogenic Right Ventricular Cardiomyopathy RQ PPARG ** ARVC Exposure to Lipogenic stimuli Disease Modeling 0 Control ARVC Control ARVC
26 Limitations and challenges of ips technology Incomplete reprogramming & Epigenetic memory Immunogenicity Cardiomyocyte Heterogeneity Early Stage cardiac phenotype.
27 Thank You Haifa, Israel Prof. Lior Gepstein s Lab. Irit Huber Amira Gepstein Gil Arbel Limor Zwi Ilanit Itzhaki Leonid Maizles Inbar Budintzky Oren Feldman Joseph Itskovitz-Eldor Michal Amit Rafael Beyar Haim Hammerman Doron Aronson Monther Bolous Hana Mandel
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