1. Biology Of Cultured Cells

2. Does Culturing Reflect Reality Culturing Deviates From In Vivo Environment –3-D matrix is disrupted (collagen, cell-cell contact) –Heterogeneity is changed –Local growth factors are removed New Environment Promotes New Properties –Progenitors are encouraged to proliferate –Differentiated cells might not have the same function as starting differentiated cells

3. Majority Of Cells Adhere On Plastic (Treated) Provided They Are Not Transformed It Was Observed That Cells Prefer –vely Charged Glass Surface Plastic (polystyrene) Is Tissue Culture Treated –With High Energy Ionizing Radiation –Electric Ion Discharge Adhesion Is Mediated By Surface Receptors And Matrix –Matrix Is Secreted By Cells, Adheres To Charged Plastic –Receptors Bind to Matrix Adhesion

4. Three Major Classes –Cell-Cell Adhesion Molecules CAMs (Ca 2+ Independent) Cadherins (Ca 2+ Dependent) Primarily Between Homologous Cells Signaling occurs –Cell-Substrate Molecules Integrins Bind to fibronectin, entactin, laminin, collagen Bind the specific motif (RGD, arginine, glycine,aspratic) Comprised of  and  unit Cell Surface Adhesion Molecules

5. 3 rd Class Is Proteoglycans –Also Binds Matrix or Other Proteoglycans –Not Via RGD Motif –Low affinity Growth Factor Receptors –May Aid Binding To Higher Affinity Receptors –No Signaling Capacity Cell Surface Adhesion Molecules

7. Spaces In Between Cells Filled With ECM –Common constituents: fibronectin, laminin, collagen, hyaluronan, proteoglycans, bound growth factors/cytokines ECM Is Dependent On Cell Types –Fibrocytes secret collagen I and fibronectin –Epithelial cells secret laminin In Most Cases Cell Lines Are Allowed To Make Their Own ECM Sometimes We Provide ECM Extracellular Matrix (ECM)

8. Cell Proliferation

9. 4 Phases –M Phase, mitosis occurs Chromatin condensation, sister chromatid separation Daughter cells –G1 Phase Progression to DNA SYNTHESIS Alternatively Go OR differentiation Restriction Points –S Phase DNA Synthesis Progression to G2 –G2 Phase Integrity of DNA Checkpoints Apoptosis is an option –DNA fragmentation, cell shrinkage, formation of small vesicles Cell Cycle

10. Environment Regulates Entry Into Cell Cycle External Growth Factors Promote Cell Proliferation –PDGF, EGF, FGF (+ve) –TGF-  (-ve) –Interact with surface receptors High Density Inhibits Proliferation (Contact Inhibition) Inside The Cell Both Positive and Negative Factors –Positive, cyclins, Growth Factor Receptor Activation –Negative, p53, Rb, Checkpoints Control Of Cell Proliferation

11. Proliferation Does NOT Promote Differentiation Differentiation Often Requires –High density –Cell-Cell Interaction –Cell-Matrix Interaction –Differentiation Factors The Above Conditions Can Be Antagonistic To Proliferation Proliferation vs Differentiation

13. 3-D Tissue Retains Its Properties Longer But Can Not Be Propagated To Overcome This Limitation –Cells Are Cultured On Matrices –Matrigel Is Commercially Available Not Perfect But Promising –Heterotypic Cultures Are Promising –Pathological Behavior Can Be Studied Tissue Retains Function Longer

14. Inability To Express In Vivo Phenotype Is Attributed To Dedifferentiation Still Not Clear If Dedifferentiation Occurs –Wrong lineage expansion is a possibility –Undifferentiated cells dominate –Absence of appropriate inducers, hormones, matrix Deadaptation vs Dedifferentiation –Deadaptation-enviroment suppresses phenotype, reversible –Dedifferentiation-conversion to primitive phenotype, irreversible Dedifferentiation

16. After 1 st Passage Primary Culture Becomes Cell Line (note  Between Finite and Continuous) By 3 rd Passage Cell Line Stabilizes Survival Of Stronger Might Not Necessarily Be The Objective Mesenchymal Cells Usually Dominate –Ex. Fibroblasts It Is Hard To Avoid Overgrowth Of Specialized Cells (Ex. Hepatic Parenchyma) Evolution Of Cell Lines

17. Approximately 10 Passages Senescence Follows –Thought To Be Due To Telomeres –Every Division Telomeres Shorten –Germ, Stem Cells Use Telomerase Transformation Is Needed If Division Will Continue Evolution Of Cell Lines

18. Finite Cell Lines Can Change To Continuous Often p53 Mutation or Deletion Occurs Overexpression Of Telomerase Transformation vs Immortalization –Transformation-additional changes in growth characteristics –Immortalization-infinite lifespan Aneuploidy Is A Characteristic Of Cont. Cell Lines –In between diploid and tetraploid –Heteroploidy is also observed Most Cells Never Become Continuous Cell Lines Continuous Cell Lines

20. Cell Plasticity and Regenerative Medicine

21. Cell Types Somatic cells share the same genome Expression profile is what determines cell type. Examples of cell types: –Cardiac Muscle cells –Fibroblasts –Neurons –Stem Cells Recent studies have shown that a small number of transcription factors can revert skin cells to Pluripotent Stem Cells –Oct4, Sox2, Nanog

22. Stem Cells Give Rise to Different Cell Types Stem Cells Can Be Totipotent or Pluripotent Totipotency is the ability to produce ALL the cell types in an organism (placenta, endoderm, mesoderm and ectoderm) –A zygote is a totipotent cell Pluripotency is the ability to produce ALL types in an organism EXCEPT placenta (endoderm, mesoderm and ectoderm)

23. Stem Cells Wikipedia, 2010

24. Stem Cells and Regeneration Stem Cells can regenerate damaged tissue For example heart Pluripotent Stem Cells (PSCs) hold great promise in regenerative medicine –A major obstacle is the danger of Stem Cells turning into tumors Scientists are working on transient expression/suppression of key genes involved in inducible PSCs –A good approach is through use of siRNA

25. Wound Healing and Regeneration Humans have little regenerative capacity primarily due to tumor suppressing genes Rb (retinoblastoma) is a key enzyme in tumor suppression. –If Rb gene is inactivated cells start acting as Stem Cells –This is a risky manipulation Arf is another important tumor suppressing gene that if turned off regeneration is observed Rb and Arf silencing was shown to result in muscle cell division and regeneration (Blau M, 2010) The wound site is a unique site where cells start dividing to repair damage tissue –Adult cells are used in this process –They start acting as ‘younger’ cells for a relatively short period of time

26. Plasticity of Fibroblasts Fibroblasts are a ubiquitous cell type Recent study showed that heart fibroblasts can be turned into cardiomyocytes (Srivastava and colleagues, 2010) Three transcription factors are needed for this transformation –Gata4 –Mef2c –Tbx5 Expression of these transcription factors is most effectively achieved using genetically engineered retroviruses Heart fibroblasts have the highest conversion efficiency into cardiomyocytes –Skin fibroblasts can also be converted with a lower efficiency

27. Reverted Fibroblasts to Cardiomyocytes Masaki, 2010  cTnT is cardiac Troponin T - a reliable marker for cardiomyocytes   MHC-GFP is a transgenic mouse with green fluorescent mature cardiomyocytes  Note Upper Right quadrant -The higher the number, the more reverted fibroblasts -Mesp1 is dispensable for troponin expression