By Deepika Chitturi BIOE 506 Spring 2009

1. Chemical and Physical Regulation of Stem Cells and Progenitor Cells: Potential for Cardiovascular Tissue Engineering (Review)Ngan F. Huang, Randall J. Lee, Song Li By Deepika Chitturi BIOE 506 Spring 2009

2. Why Cardiovascular Tissue Engineering? • Leading Cause of Mortality (every 34 sec) • Expensive ($250 billion) • Myocardial Infarction (MI aka heart-attacks) • Coronary Artery Occlusion • Cardiomyocyte Cell Death • Non-generation • Formation of Scar Tissue • Dilation of Chamber Cavities • Aneurysmal Thinning of Walls • REDUCED PUMPING CAPACITY • Driving Force: Shortage of Donors

3. Potential Stem & Progenitor Cells • MSCs: Mesenchymal Stem Cells • HSCs: Hematopoietic Stem Cells • EPCs: Endothelial Precursor Cells • ESCs: Embryonic Stem Cells • Skeletal Myoblasts • Resident Cardiac Stem Cells

4. Perfect Tissue Engineered Construct • CELL SOURCE • SOLUBLE CHEMICAL FACTORS • EXTRACELLULAR MATRIX (ECM)

5. Cardiovascular Tissue Engineering (I) • Cell Source • Embryonic Stem Cells • Adult Stem Cells • Soluble Chemical Factors • VEGF (ESCs, HSCs, EPCs) • TGF-β(ESCs, MSCs, HSCs, EPCs) • BMP (ESCs) • 5-azacytidine (MSCs) • FGF (ESCs, HSCs, EPCs) • IGF (HSCs, EPCs)

6. Cardiovascular Tissue Engineering (II) • Extracellular Matrix • Natural Polymers • Matrigel: In vivo injection for MI, ESC differentiation • Collagen: In vivo injection for MI, Vascular grafts • Hyalinuric Acid: Vascular grafts • Alginate: ESC differentiation • Fibrin: In vivo injection for MI, Vascular conduits • Decellularized Vessel: Vascular conduits • Synthetic Polymers • Poly-L-lactic Acid (PLLA): ESC differentiation • Poly-lactic-co-glycolic acid (PLGA): ESC differentiation • Polyglycotic Acid (PGA): Vascular grafts • Peptide Nanofibers: In vivo injection for MI • Poly-diol-citrates and Poly-glycerol-sebacate: General tissue engineering

7. Extracellular Matrix Matrigel Angiogenesis PLLA Angiogenesis Dr. Vasif Harsirci- Middle East Technical University (Biomedical Unit) Effects of Cordycepsmilitaris extract on angiogenesis and tumor growth1Hwa-seung YOO, Jang-woo SHIN2, Jung-hyo CHO, Chang-gue SON, Yeon-weol LEE, Sang-yong PARK3, Chong-kwan CHO4 Department of East-West Cancer Center, College of Oriental Medicine, Daejeon University, Daejeon 301-724;

8. Role of Matrix Materials for Structural Support • hESCs cultured in porous PLGA/PLLA scaffolds coated with Matrigel or Fibronectin vs. Matrigel alone or fibronectin-coated dishes (Levenberg et al) • 3-D polymer structure promoted differentiation (neural tissue, cartilage, liver and blood vessels) • Formation of 3-D blood vessels • Fibronectin-coated dishes: • Failure to organize into 3-D structure • Matrigel: • Organization into 3-D structure • No cell differentiation • Conclusion: • Large inter-connected pores: cell colonization • Pores smaller than 100 nm: limit diffusion of nutrients and gases • 3-D: great surface area, higher expression of integrins

9. Role of Matrix Topography and Rigidity • Topography: Cell Organization, alignment and differentiation • Nano-scale and micro-scale matrix topography affects organization and differentiation of stem cells • hMSCs undergo skeletal reorganization and orient themselves in the direction of microgrooves and nano-fibers (Patel et al) • Stiffness/Rigidity: Cells tend to migrate toward more-rigid surfaces and cells on soft matrix have a low rate of DNA synthesis and growth (Engler et al) • Assembly of focal adhesions and contractile cytoskeleton structure depend on rigidity

10. Cardiovascular Tissue Engineering Models • In vitro differentiation method: engineering constructs with structural and functional properties as native tissues before transplantation • In situ method: relies on host environment to remodel the chemical and physical environment for cell growth and function • Ex vivo approach: excision of native tissues and remodeling them in culture

11. Cardiovascular Tissue Engineering Proposed Models • Injectable Stem Cells and Progenitor Cells for in situ cardiac tissue engineering • Vascular Conduits

12. Injectable Stem Cells and Progenitor Cells for in situ cardiac tissue engineering • Delivery modes for myocardial constructs: • Cardiac patching • Cell Injection • Cell-polymer injection • Less invasive than solid scaffolds • Adopt shape and form of host environment • Delivery vehicles (with cells and GFs) • Polymers: Collagen I, Matrigel, Fibrin, Alginate and Peptide Nanofibers

13. Injectable delivery of Polymers • Collagen I, Matrigel and Fibrin • Higher capillary density than saline control treatment • Migration of vascular cells into infarcted region for neovascularization • Fibrin + MSCs (Huang et al) • Promotes angiogenesis • ESCs + Matrigel (Kofidis et al) • Greater improvements in contractility after 2 weeks • Rat bone marrow mononuclear cells (MNCs) + Fibrin (Ryu et al) • Enhanced neovascularization • Development of larger vessels • Extensive tissue regeneration • Graft survival: 8 weeks

14. Treatment using Stem and Progenitor Cells alone • TGF-β-treated CD117+ rat MNCs (Li et al) • Differentiation into myogenic lineage • Enhanced vascular density • Retrovirallytransduced Akt1-overexpressing MSCs (Mangi et al, Laflamme et al) • Reduced intramyocardial inflammation • 80% of lost myocardial volume regeneration • Normal systolic and diastolic functions restoration • Cardiac enriched hESCs in athymic rats (Laflamme et al) • Cardiomyocyte growth • No teratomas • 7-fold increase in graft size in 4 weeks • Potential regeneration of human myocardium in rat heart

15. Vascular Conduits • Goal: To create functional conduit as a bypass graft (small, non-thrombogenic, native mechanical properties) • Limitations to vein grafts: • Availability • 35% 10-year failure • Synthetic Vascular Grafts: • Poly-ethylene-terephthalate • Expanded poly-tetrafluoroethylene • Polyurethane • Limitation: • Inside diameter larger than 5 mm • Frequent thrombosis and occlusions in smaller grafts

16. Vascular Conduits—Proposed Models • ECs + SMCs in a tubular PGA porous scaffold (Niklason et al) • In vivo implantation: patent for 2 weeks; development of histological features consistent with vascular structures • EPC-seeded grafts (Kaushal et al) • Remained patent for more than 130 days • Acellular control grafts occluded in 15 days • Vessel-like characteristics: contractility and nitric-oxide mediated vascular relaxation • EPCs derived from umbilical cord blood using 3D porous polyurethane tubular scaffolds in a biomimetic flow system (Schmidt et al) • In 12 days, EPCs lined lumen of VGs and formed endothelial morphology

17. Vascular Conduits—Proposed Models • MSC seeded nanofibrous vascular grafts (Hashi et al) • Patent for at least 8 weeks • Synthesis and organization of collagen and elastin • EC monolayer formed on lumen surfaces • SMCs were recruited and formed

18. Conclusion • Understanding the effect of chemical and physical cues for regulation of stem-cell survival, differentiation, organization and morphogenesis into tissue-like structures: most important!! • Cardiovascular repair, Cardiac therapies after MI and engineering of vascular conduits