Biology of cultured cells
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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

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Does culturing reflect reality
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
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
Cell Surface Adhesion Molecules

  • Three Major Classes

    • Cell-Cell Adhesion Molecules

      • CAMs (Ca2+ Independent)

      • Cadherins (Ca2+ 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 molecules1
Cell Surface Adhesion Molecules

  • 3rd 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


Extracellular matrix ecm
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 cycle
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
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 vs Differentiation

  • 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


Tissue retains function longer
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
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
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 lines1
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
Continuous Cell Lines

  • 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 Plasticity and Regenerative Medicine


Cell types
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


Stem cells give rise to different cell types
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)


Biology of cultured cells

Stem Cells

Wikipedia, 2010


Stem cells and regeneration
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
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
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
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