Molecular control of cell fate decision in pluripotent and adult stem cells: from basic science toward therapy Gabriella Minchiotti INBB, Roma 24 Ottobre 2014 Convergent pathways in embryogenesis, oncogenesis and tissue regeneration Cancer Stem Cells Embryonic Stem Cells ONCOGENESIS EMBRYOGENESIS Primary Tumor Zygote Blastocyst Gastrula Adult Stem Cells Tissue Regeneration Tissue Regeneration Adult Stem Cell Activation/Differantiation Metastasis Mechanisms of cell lineage specification in mammals TAC TACs- Transit Amplifying Cells Adapted S. Pece et al., BBA 2011 Role of local environmental cues in defining cell-type identity Stem cell Modified from Scadden D., Nature 2006 Signaling pathways involved in cell fate specification Blastocyst Growth of ICM cells Germ layers specification ES cells Follistatin cerberus EGF-CFC cripto Activins Nodal Nodal-related Vg1 TGF-1, 2, 3 BMP2 BMP4 BMP7 DDF6 Munoz‐Sanjuan and Brivanlou, 2002 Cripto redirects the neural fate of ESCs + Cripto - Cripto MF20 MF20 + Cripto - Cripto III tubulin III tubulin Parisi et al., JCB 2003 Genetic or pharmacological inhibition of Cripto in mouse ESCs cells improves functional integration of ESCs and blocks tumor formation in Parkinsonian rats % change in rotational behaviour wild-type ESCs Cripto -/- ESCs Parish, et al., Stem Cells, 2005 Lonardo, et al., Stem Cells, 2010 Cripto controls cell lineage specification in ESCs Therapeutic target Parkinsons’ disease Parish et al., Stem Cells, 2005 Cripto HIF-1 APJ/Apelin APJ-Cerberus-Baf60C Lonardo et al., Stem Cells, 2010 Patent- EP09166967 Bianco et al., Am. J. Pathology, 2009 D’Aniello et al., Circ Res, 2010 D’Aniello et al., Cardiovasc. Res., 2013 Cripto regulates cell lineage commitment in pluripotent stem cells Pluripotent CRIPTO Adapted S. Pece et al., BBA 2011 ADULT STEM CELLs?? Common pathways in embryonic and adult stem cells Embryonic Stem Cells Cripto Adult Stem Cells Cripto Skeletal Muscle Regeneration Skeletal muscle regeneration Myogenic satellite cells: physiology to molecular biology, T. J. Hawke, 2001 Sequential expression of myogenic transcription factors The Skeletal Muscle Satellite Cell: The Stem Cell That Came inFrom the Cold. Peter S. Zammit et al., 2006 Cripto is expressed in myoblasts and inflammatory cells in skeletal muscle regeneration Guardiola et al., PNAS 2012 Cripto is expressed in myogenic precursor cells in isolated myofibers Cripto Pax7 DAPI MyoD Cripto BF B C D E F G H I J M N O T24 T0 A L T48 K Guardiola et al., PNAS 2012 Cripto promotes satellite cell lineage progression and proliferation Isolated myofibers Model of satellite cell self-renewal Number of positive cells (%) * ** ** ** * * Control Zammit P S et al. J Cell Biol 2004 ** sCripto T48 Control sCripto Control T72 sCripto 0‐96h sCripto 0‐ 48h T96 Committed myoblasts to differentiation (Pax7‐/MyoD+) Activated/proliferating satellite cells (Pax7+/Myod+) Quiescent satellite cells (Pax7+/MyoD‐) Guardiola et al., PNAS 2012 Cripto promotes satellite cell myogenic commitment antagonizing Myostatin Control Myostatin + Cripto Myostatin Committed myoblasts (Pax7‐/MyoD+) Activated/proliferating satellite cells (Pax7+/Myod+) Quiescent satellite cells (Pax7+/MyoD‐) Guardiola et al., PNAS 2012 CONDITIONAL SATELLITE CELL –SPECIFIC CRIPTO KO Tg:Pax7-CreERT2::CriptoLoxP/- CONDITIONAL SATELLITE CELL –SPECIFIC CRIPTO GOF Tg:Pax7-CreERT2::CriptoGOF Satellite cell-specific cripto ablation affects muscle regeneration Tg:Pax7-CreERT2::CriptoLoxP/- Guardiola et al., PNAS 2012 Impacts of Genetic modulation of Cripto signaling in the satellite cell compartment on skeletal muscle regeneration Toward Pharmacological modulation of Cripto signaling for skeletal muscle regeneration The Cell maker The system executes Protocols for targeted differentiation of ES cells Screening of compounds libraries Complete sterility Advantage/benefits High standard reliability, performance and flexibility Low user intervention Contamination risk reduction 96-channel head disposable pipetting tips Method standardization Up to 4000 single compuonds simultaneously screened Co developed with Hamilton Robotics + Cell-based Phenotypic Screening: Cell Proliferation (36hrs) Cell Colony Phenotype (5days) Small molecules • Metabolic Intermediates • HDAC Inhibitors • FDA-approved drugs Casalino et al., JMCB, 2011 Casalino et al., Molecular Biotechnology, 2011 Franci et al., Biol. Open, 2013 Comes et al., Stem Cell Reports, 2013 Targeted Differentiation (10-13 days) neurons cardiomyocytes L Proline –induced cells (PiCs): a novel metastable state of pluripotent stem cells ESCs PiCs Sensitivity to trypsin digestion ‐ + Alkaline phoshatase activity + ‐ In vitro cardiac and neural differentiation + + Teratoma formation and blastocyst colonization + + PiCs Control L-proline BF EGFP PiCs = Proline induced Cells E 11.5 Casalino, Comes et al., JMCB, 2011 E 11.5 L-Pro induces remodeling of the ESC transcriptome Comes et al., Stem Cell Reports 2013 L-Proline -induced cytoskeletal rearrangements in ESCs ESCs PiCs 50 µm 50 µm PiCs 50 µm 50 µm 50 µm 50 µm 50 µm 30 µm 20 µm Comes et al., Stem Cell Reports 2013 L-Pro induces a motile phenotype in ESCs Control ESCs L- proline -treated ESCs From Day 3 to Day 4 (images were collected every 5 minutes/ 20x objective) ESCs ** PiCs 100 80 60 * 40 20 0 1-15% * migration invasion ** 150 100 50 0 0 ESCs 0 100 PiCs PiCs ESCs 15% FBS 200 1x106 ESCs or PiCs injected into tail veins; mice were sacrificed 4 weeks after injection SDF-1 (ng/ml) EGFP+ PiCs Cell Migration (cells per field) 120 B16-BL6 140 Cell Migration / Invasion (cells per field) PiCs are invasive and metastatic pluripotent stem cells Comes et al., Stem Cell Reports 2013 L-Proline induces a fully reversible EMT –like transition in embryonic stem cells: embryonic stem to Mesenchymal Transition (esMT) B Control 1 1 100 500 Relative Expression 3 5 GADPH -36 E Slug kDa GADPH -36 E-cad 0 Control 1 Control 3 4 5 ++ L-Pro L-Pro(day) (day) day 3 E-CAD/Hoechst day 5 Relative E-CAD Level -120 Comes et al., Stem Cell Reports 2013 kDa -135 Snail E-CAD 0 5 -50 L-Pro(day) (day) ++ L-Pro 4 4 T + L-Pro (day) Control 3 N-CAD 10 5 N-cad 100 4 Vim + L-Pro (day) N-cad T 3 T 1000 2 MMP-2 Control Relative Expression A 1 2 Relative Expression + L-Pro compacted stem cells scattered stem cells L-Proline esMT MesT E-cadherin Vitamin C E-cadherin teratoma invasiveness Vitamin C is a key cofactor of the reactions driven by histone demethylases of the JMJ family Vitamin C improves cell riprogramming (Shi et al., Cell Stem Cell 2009) by modulating H3K9 and H3K36 methylation (Chen J. et al Nature Genet., 2012) L-Proline is a genome-wide inducer of H3K9 and H3K36 methylation Comes et al., Stem Cell Reports 2013 L-Pro triggers an esMT reminiscent of the EMT that occurs at the invasive front of the tumor, and contributes to the acquisition of cell plasticity and invasiveness. L-Pro–induced esMT is fully reversible (MesT) and is accompanied by a global remodeling of H3K9 and H3K36 methylation status compacted stem cells scattered stem cells L-Proline esMT MesT E-cadherin Vitamin C E-cadherin teratoma invasiveness Lab members Past members Salvatore Iaconis Stefania Comes Nicola Laprano External collaborations Ombretta Guardiola Annalisa Fico Carolina Prezioso Alessandro Fiorenzano Cristina D’Aniello Gennaro Andolfi Giorgio Scita, IFOM, Milan Claudia Angelini, Institute for Applied Mathematics, CNR, Naples Silvia Brunelli, HSR, Milan Michael Shen, Columbia University, NY Sharhaghim Tajbakhsh, Pasteur Institute, Paris Ann Zeuner, Ruggero de Maria, ISS, Rome TIGEM Bioinformatic Core Facility,, Naples Dror Seliktar, Technion, Haifa, Israel Thanks to IGB collaborations Eduardo J. Patriarca Maria Matarazzo Dario De Cesare Laura Casalino IGB Facilities Integrated Microscopy Animal House FACS NGS EMBOWORKSHOP ON StemCell Mechanobiology in Development and Disease October 18-21, 2015 Capri, Naples Yohanns Bellaïche, France Françoise Brochard-Wyart, France Chen Christofer S., USA Giulio Cossu, UK George Q. Daley, USA Carl-Philipp Heisenberg, Austria Donald E. Ingber, USA Benoît Ladoux, France David A. Lee, UK Ohad Medalla, Switzerland Christine Mummery, The Netherlands Paolo Netti, Italy Graziella Pellegrini, Italy Stefano Piccolo, Italy Mattieu Piel, France Giorgio Scita, Italy Dror Sellktar, Israel GV Shivashankar, Italy Craig A. Simons, Canada Molly Stevens, UK Viola Vogel, Switzerland Fiona Watt, UK Gabriella Minchiotti, Institute of Genetics and Biophysics ‘A. Buzzati Traverso’, (IGB), Naples, Italy Paolo A. Netti, Italian Institute of Technology (IIT), Naples, Italy Viola Vogel, Laboratory of Applied Mechanobiology, ETH, Zürich, Switzerland The IGB Meeting Coordinators Maria R. Matarazzo, Maria G. Miano Workshop Secretariat Anna Maria Aliperti, Federica Staempfli
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