Stem cells and tissue engineering
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This document discusses regenerative medicine and tissue engineering. It outlines examples of regeneration in nature and clinical needs where regeneration could help such as heart disease and bone fractures. Stem cells are described as a potential cell source along with factors like growth factors and scaffold materials. Challenges in tissue engineering like optimal cell delivery and scaffold design are covered. Cardiovascular applications are discussed in depth as a promising target for regenerative approaches.
Stem cells and tissue engineering
- 1. Aaron Maki April 24, 2008
- 2. Regeneration in Nature Outstanding Examples Planarian Crayfish Embryos Inverse Relationship Increase complexity Decrease regenerative ability
- 3. Regeneration in Humans High Moderate Low
- 4. Clinical Needs Cardiovascular Myocardial infarction Stroke Bone Non-union fractures Tumor resections Nervous Spinal Cord Injury Degenerative diseases
- 5. Stem Cells Long-term self-renewal Clonogenic Environment-dependent differentiation
- 6. Tissue Engineering Repair/replace damaged tissues Enhance natural regeneration Cell Source Embryonic stem cells Adult stem cells Progenitor cells Signals Growth factors Drugs Mechanical forces ECM Metals Ceramics Synthetic polymers Natural polymers
- 7. Important Variables Delivery Cell Suspensions Tissue-like constructs (scaffolds) Chemical properties Growth factors Degradation particles ECM surface Physical properties Structure Topography Rigidity Mechanical Loading Modify Cell Behavior Survival Organization Migration Proliferation Differentiation Optimize Cellular Response
- 8. Stem and Progenitor Cells Isolation/Identification Signature of cell surface markers Surface adherence Transcription factors Classifications Embryonic Stem Cells Adult Stem Cells Induced Pluripotent Stem Cells
- 9. Embryonic Stem Cells Highest level of pluripotency All somatic cell types Unlimited self-renewal Enhanced telomerase activity Markers Oct-4, Nanog, SSEA-3/4 Limitations Teratoma Formation Animal pathogens Immune Response Ethics Strengths
- 10. Potential Solutions Teratoma Formation Pre-differentiate cells in culture then insert Animal pathogens Feeder-free culture conditions (Matrigel) Immune Response Somatic cell nuclear transfer Universalize DNA Ethics
- 11. Adult Stem Cells Strengths Ethics, not controversial Immune-privileged Allogenic, xenogenic transplantation Many sources Most somatic tissues Limitations Differentiation Capacity? Self-renewal? Rarity among somatic cells
- 12. Potential Solutions Differentiation Capacity Mimic stem cell niche Limited Self-renewal Gene therapy Limited availability Fluorescence-activated cell sorting Adherence Heterogenous population works better clinically
- 13. Mesenchymal Stem Cells Easy isolation, high expansion, reproducible
- 14. Hematopoietic Stem Cells Best-studied, used clinically for 30+ years
- 15. Induced Pluripotent Stem Cells Strengths Patient DNA match Similar to embryonic stem cells? Limitations Same genetic pre-dispositions Viral gene delivery mechanism
- 16. Potential Solutions Same genetic pre-dispositions Gene therapy in culture Viral gene delivery mechanism Polymer, liposome, controlled-release Use of known onco-genes Try other combinations
- 17. Soluble Chemical Factors Transduce signals Cell type-dependent Differentiation stage-dependent Timing is critical Dose-dependence Growth Survival Motility Differentiation
- 18. Scaffold purpose Temporary structural support Maintain shape Cellular microenvironment High surface area/volume ECM secretion Integrin expression Facilitate cell migration Surface coating Structural
- 19. Ideal Extracellular Matrix 3-dimensional Cross-linked Porous Biodegradable Proper surface chemistry Matching mechanical strength Biocompatible Promotes natural healing Accessibility Commercial Feasibility Modulate Properties Physical, Chemical Customize scaffold Appropriate Trade-offs Tissue Disease condition
- 20. “Natural” Materials Polymers Collagen Laminin Fibrin Matrigel Decellularized matrix Ceramics Hydroxyapatite Calcium phosphate Bioglass Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart. Ott, et al. Nat Med. 2008 Feb;14(2):213
- 21. Important scaffold variables Surface chemistry Matrix topography Cell organization, alignment Fiber alignment -> tissue development Rigidity 5-23 kPa Porosity Large interconnected small disconnected
- 22. Mechanical Forces Flow-induced shear stress Laminar blood flow Rhythmic pulses Uniaxial, Equiaxial stretch Magnitude Frequency Mechanotransduction Conversion of a mechanical stimulus into a biochemical response
- 23. Flow-induced shear stress 2D parallel plate flow chamber Hemodynamic force Laminar flow Pulsatile component 3D matrix Interstitial flow Bone: oscillating Cell-type specific
- 24. Models for Tissue Engineering In vitro differentiation Construct tissues outside body before transplantation Ultimate goal Most economical Least waiting time In situ methodology Host remodeling of environment Ex vivo approach Excision and remodeling in culture Combine physical and chemical factors Optimize stem cell differentiation and organization
- 25. Delivery Methods Injectable stem cells Cells or cell-polymer mix Less invasive Adopt shape of environment Controlled growth factor release Solid scaffold manufacturing Computer-aided design Match defect shape
- 26. Cardiovascular Tissue Engineering Heals poorly after damage (non-functional scar tissue) Myocardial infarction 60% survival rate after 2 years >40% tissue death requires transplantation More patients than organ donors Heart attack and strokes First and third leading causes of death Patient often otherwise healthy
- 27. Current interventions Balloon angioplasty Expanded at plaque site, contents collected Vascular stent Deploy to maintain opening Saphenous vein graft Gold Standard Form new conduit, bypass blockage All interventions ultimately fail 10 years maximum lifetime
- 28. Cardiovascular Tissue Engineering Cell Source Embryonic stem cells Mesenchymal stem cells Endothelial progenitor cells Resident Cardiac SCs Signals VEGF TGF-β FGF BMP PDGF Shear stress Axial strain ECM Matrigel Collagen Alginate Fibrin Decellularized Tissue PLA PGA
- 29. Clinical Questions What cell source do you use? How should cells be delivered? What cells within that pool are beneficial? How many cells do you need? When should you deliver the cells? What type of scaffold should be used? These answers all depend on each other
- 30. Very sensitive to methodology! 2 nearly identical clinical trials, opposite results Autologous Stem cell Transplantation in Acute Myocardial Infarction (ASTAMI) Reinfusion of Enriched Progenitor cells And Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) Same inclusion criteria Same cell source (Bone marrow aspirates) Same delivery mechanism (intracoronary infusion) Same timing of delivery SIMILAR cell preparation methods Seeger et al. European Heart Journal 28:766-772 (2007)
- 31. Cell preparation comparison Bone marrow aspirates diluted with 0.9% NaCl (1:5) Mononuclear cells isolated on Lymphoprep™ gradient 800rcf 20 min Washed 3 x 45 mL saline + 1% autologous plasma (250rcf) Stored overnight 4°C saline + 20 autologous plasma Bone marrow aspirates diluted with 0.9% NaCl (1:5) Mononuclear cells isolated on Ficoll™ gradient 800rcf 20 min Washed 3 x 45mL PBS (800rcf) Stored overnight room temperature in 10 + 20% autologous serum Courtesy of Dr. Tor Jensen
- 32. Future Directions Standardization Central cell processing facilities Protocols Improved antimicrobial methods Allergies Synthetic biology Natural materials made synthetically, economically
- 33. Long-term: “clinical-grade” cell lines Animal-substance free conditions Human feeder cells, chemically-defined media Feeder-free culture No immune rejection, no immunosuppressive drugs Somatic cell nuclear transfer Genetic engineering, reprogramming Goals: understand normal/disease development, then repair/replace diseased organs and vice versa Tissue engineering approach ex vivo, in situ for now In vitro for the future?
- 34. Summary Right combination of cell, scaffold, and factors depends on clinical problem Extensive physician/scientist/engineering collaboration is vital to success Tissue engineering is leveraging our knowledge of cell biology and materials science to promote tissue regeneration where the natural process is not enough Stem cells are an excellent tool for this task