Download Biology Of Cultured Cells Chpt. 3

Biology Of Cultured Cells
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
Adhesion
• 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
Cell Surface Adhesion Molecules
• Three Major Classes
– Cell-Cell Adhesion Molecules
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CAMs (Ca2+ Independent)
Cadherins (Ca2+ Dependent)
Primarily Between Homologous Cells
Signaling occurs
– Cell-Substrate Molecules
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Integrins
Bind to fibronectin, entactin, laminin, collagen
Bind the specific motif (RGD, arginine, glycine,aspratic)
Comprised of  and  unit
Cell Surface Adhesion Molecules
• 3rd Class Is Proteoglycans
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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
Extracellular Matrix (ECM)
• 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
Cell Proliferation
Cell Cycle
• 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
Control Of Cell Proliferation
• 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
Proliferation vs Differentiation
• Proliferation Does NOT Promote Differentiation
• Differentiation Often Requires
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High density
Cell-Cell Interaction
Cell-Matrix Interaction
Differentiation Factors
• The Above Conditions Can Be Antagonistic To
Proliferation
Tissue Retains Function Longer
• 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
Dedifferentiation
• 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
Evolution Of Cell Lines
• After 1st Passage Primary Culture Becomes Cell
Line (note  Between Finite and Continuous)
• By 3rd 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
• 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
Continuous Cell Lines
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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
Cell Plasticity and
Regenerative Medicine
Cell Types
• Somatic cells share the same genome
• Expression profile is what determines cell
type. Examples of cell types:
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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
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)
Stem Cells
Wikipedia, 2010
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
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
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
Reverted Fibroblasts to Cardiomyocytes
 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
Masaki, 2010