How to Make a Heart: Towards Pluripotent Cell-Based Heart Stem Cell Therapeutics

1. How to Make a Heart: Towards Pluripotent Cell-Based Heart Stem Cell Therapeutics ACI 2011 BCIS Meeting London Hilton Metropole London, UK January 28, 2011 Kenneth R. Chien MD, PhD MGH Cardiovascular Research Center Department of Stem Cell and Regenerative Biology Harvard University Harvard Stem Cell Institute • Islet-1 heart progenitor story • Heart Parts: Engineering patch of mature heart muscle tissue from ES cells • Human master Islet-1 progenitors: Human cardiogenesis, congenital heart disease , and regenerative therapeutics • Laser technology devices for cell based therapy

2. The Unmet Clinical Need: Growing Heart Muscle

3. Regeneration Next: Towards Heart Progenitor Derived Heart Parts Hansson, Lindsay, and Chien, Cell Stem Cell, In Press, September, 2009

4. The Meltonian Stem Cell Era: Directed Differentiation of Pluripotent Stem Cells J Embryol Exp Morphol. 1985 The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. • Diversification • Scalability • Maturation • Coordination • Durability

5. How to Make a Heart Ventricular Cardiac Muscle Cell Fate Map: Towards Human Heart Stem Cell Models and Therapeutics • Laugwitz et al, Nature, 2005 • Moretti et al Cell, 2006 • Qyang et al Cell Stem Cell, 2007 • Martin-Puig et al, Cell Stem Cell, 2008 • Chien, Nature, 2008 • Chien, et al, Science, 2008 • Bu et al, Nature, 2009 • Domian et al, Science, 2009 • Hansson et al, Cell Stem Cell, 2009 ES/iPS Pluripotent Stem Cells Multipotent Iwlet Heart Progenitor Committed Ventricular Progenitor Differentiated Ventricular Cell Mature Ventriuclar Cell Durable Ventricular Muscle Tissue

6. Towards defining the heart cell lineage map Bipotent Progenitor Differentiated Progeny Smooth Muscle Cell Multipotent Isl1 Cardiovascular Progenitor ? SM-MHC Wnt/b-cat Right * Ventricular Cardiomyocyte Wnt/b-cat Mlc2-v MHC Isl-1 Nkx2.5 Atrioventricular Nodal Cell Atrial Cardiomyocyte Isl-1 Flk-1 Nkx2.5 Common Primordial Cardiovascular Progenitor Cardiac Mesodermal Precursor Mesodermal Precursor SLN MHC ? Sinoatrial Nodal Cell Endothelial Cell CD31 vWf Bry Bry Flk-1 Smooth Muscle Cell SM-MHC Primary Heart Field Cardiovascular Progenitor Left * Ventricular Cardiomyocyte Nkx2.5 Others ? Mlc2-v Nkx2.5 MHC

7. Coordinated Contractility of a Syncitium of Murine ES cell derived Islet-1 Progenitors Driven into the Cardiac Lineage

8. Cardiac Progenitor Cells Form a Thin Film of Beating Cardiac Muscle

9. Making a Ventricular Heart Muscle Patch: Towards Regenerative Cardiovascular Medicine Domian et al, Science, 2009

10. Islet Heart Progenitors in the Human Heart • Hansson, Lindsay, and Chien, • Cell Stem Cell, October, 2009 Bu et al, Nature, 2009

11. Master Human Islet Heart Progenitors from ES Cells Can Make Any Cardiovascular Heart Cell Type Master ISL1 heart progenitors

12. A Stem Cell Paradigm for Human Cardiogenesis & Congenital Heart Disease • TBX1 • NKX2.5 • TBX20 Hansson, Lindsay, and Chien, Cell Stem Cell, October, 2009

13. Loeys-Dietz syndrome Malformation of several organs, including the cardiovascular system - arterial tortuosity - diffuse aneurysms - rupture / death young age - congenital heart disease (PDA, BAV, ASD) Caused by mutations in TGFRB1 (ALK5) or TGFRB2 Loeys et al., Nature Genetics, 2005

14. Analysis of SHF progenitor cells carrying TGFBR mutations causing Loeys-Dietz syndrome iPS derivation EB differentiation Plate & culture under ISL1+ promoting conditions + / - Alk5 inhibitor LD syndrome iPS cells display a >4 fold increase in the induction of ISL+ progenitors +/- Alk5 inhibitor vs control

15. A Islet Progenitor Fate Map for Regenerative Cardiovascular Medicine TGF-beta - Intermediates committed to CV lineages Multipotent Isl1+ cardiovascular progenitors Mesodermal precursors Cardiomyocytes CM Progenitors differentiation prespecification Smooth muscle Wnt3a Islet/ Wnt3a ? Endothelial cells Cardiac mesenchymal cells • Diversification (Islet) • Scalability (Wnt) • Maturation (TGF beta Pathway) • Coordination (?)

16. The “Apollo” Mission of Cardiac Regenerative Medicine Heart Muscle Tissue Engineering In vivo delivery Vascularization Grafting/Rejection Side effects/ arrhythmogenesis Efficacy endpoints

17. General Pathway for Cell-based Regenerative Medicine

18. Lasers in Medicine Ophthalmology Dermatology Plastic Surgery Cardiology Oncology Vascular Medicine Radiology

19. Advantages of Lasers in Medicine • Finely focused laser beams offer precision over scalpels • Hemostasis • Miniaturization of laser • Adaptability to different needs • Many types of lasers with properties tunable to specific needs

20. Liver Day 0 Day 1

21. Liver Day 7 Day 4

22. Skeletal muscle regeneration CD-1 Mice • Lasers can create finely focused areas of injury • Skeletal muscle regenerates quickly with a minimum of fibrosis

23. Human, Human, and Human:Towards Human Heart Parts and Heart Models • The checkpoints that control the expansion of ventricular progenitors? • Promoting the in vivo survival and grafting of CVP derived heart patches and in the in vivo context • In vivo delivery of CVPs—miniaturized endoscopic delivery systems • Engineering other “Heart Parts”---Coronary Arteriogenesis • Drug discovery/safety in human models systems • CHD and adult monogenic CV human disease models