sabrina’jedlicka’ interactionsofneuronsand materialsinbios21/pdf/fall2015/jedlicka...mimic the...
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Interactions of Neurons and Materials
Sabrina Jedlicka
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Neurological Disorders § Over 600 known neurological disorders
ú Diseases of the CNS and PNS Epilepsy Alzheimer’s Parkinson’s Etc.
ú Diseases that attack the nervous system Infections Cancers
ú Physical Injury ú Stroke ú Sensory
§ For most, treatment options are extremely limited § The disease/disorder mechanism is highly varied, as
is the prognosis.
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Primary Goals
§ Analyze the function of the nervous system § Develop methods to restore damaged neurological functions
§ Create artificial neuronal systems
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How to grow neuronal cells in culture…From proliferation to differentiation
There is a subset of bioengineering that focuses on developing biomaterials for integration with neural systems, either in vivo or in vitro.
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Cell-‐Material Interactions Neural Stem
Cells
Surface-Induced Chemotransduction
Cellular Mechanotransduction
Cellular Biomechanics
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Definitions
§ Cellular Mechanotransductionú The mechanism by which cells convert mechanical signals in biochemical responses
§ Cellular Mechanobiologyú Characterization of cell mechanics
§ Cellular Surface Induced Chemotransductionú The mechanism by which cells respond to surface-bound signals (such as ECM proteins)
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Why does chemo-mechanotransduction matter?
§ Cell Proliferationú Disease Statesú Propagation of sufficient cell numbers for therapeutics
§ Cell Communicationú Force translation via cell-cell contactsú Downstream biochemical signaling
§ Cell Differentiationú Development of useful cell-based models for research
ú Differentiation of cells into more mature phenotypes for transplantation
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Research History
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Research History
Engler, A. J., Sen, S., Sweeney, H. L., & Discher, D. E. (2006). Matrix elasticity directs stem cell lineage specification. Cell, 126(4), 677-‐689.
� Mesenchymal stem cells differentiate based on substrate elasticity (Engler et al., 2006)
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Developing Bio-inspired Materials…
Ingber, D. E. (2006). Cellular mechanotransduction: Putting all the pieces together again. Faseb Journal, 20(7), 811-‐827
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C17.2 Neural Stem Cells
§ Neuronal differentiation is controlled via serum withdrawal (starvation), which usually results in a homogeneous population of neurons
But…
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Controlling Differentiation
§ In vivo processes are highly coordinated and elegant.
§ Some of the proteins involved in asymmetric division are known (Example: Numb), but some of their roles are not clearly defined
§ However, in a lab setting, when you are trying to regrow neurons for therapeutic purposes – you often start with stem cells in a petri dish, which does not have the elegant coordination of signals to trigger cell changes at the right time and place.
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Goals: Enhancing Differentiation
Controlling the chemomechanical environment may allow us to provide the right signals to stimulate NSCs to become post-‐mitotic neurons of phenotype X for treatment of a certain disease. Alternatively, it will provide a means to produce new model system to study novel drugs And lots of other applications!
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NSCs and Mechanosensing: The Research QuestionHow do material cues impact differentiation of NSCs?
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Materials Inspiration Activity Independent Mechanisms
Stem CellDifferentiation Migration
Maturation
“Hardwired” or Environmentally Influenced
Environmental Factors Influencing Activity Independent Mechanisms
ECM Composition and Concentration
Growth Factors
Morphogens
Cell-Cell Interactions
Mechanical Environment
We can control or mimic many of these factors
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Mimic the extracellular environment during early corticogenesis
Diverse Neuronal Population
ECM Is Critical for Appropriate Cell Differentiation and Migration
Materials Design
Laminin Fibronectin Collagen
Shc
GRB2/mSos
Ras
Erk
C-Fos/c -Jun
FAK
Src Cas
Rac
PAK
Rho
Jnk
P1-3K
Neuronal cytoarchitecture
SCG10
Cdc42
Cell polarity
Lamellipodia formation and
lamellipodia/filopodiaextension
Gene TranscriptionAxonal
guidance
FGF pathways
Cadherincontaining cadherinsjunctions
Laminin Fibronectin Collagen
Shc
GRB2/mSos
Ras
Erk
C-Fos/c -Jun
FAK
Src Cas
Rac
PAK
Rho
Jnk
P1-3K
Neuronal cytoarchitecture
SCG10
Laminin Fibronectin Collagen
Shc
GRB2/mSos
Ras
Erk
C-Fos/c -Jun
FAK
Src Cas
Rac
PAK
Rho
Jnk
P1-3K
Neuronal cytoarchitecture
SCG10
Cdc42
Cell polarity
Lamellipodia formation and
lamellipodia/filopodiaextension
Gene TranscriptionAxonal
guidance
FGF pathways
Cadherincontaining cadherinsjunctions
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Examine Cellular Response To Material Surfaces
Investigate Cell Type Diversity Changes
Flow Cytometry Analysis of Cell Types on Control Surfaces
Neurons
β-tubulin III
Astrocytes
GFAP
25-30% 10-20%
Cell Type Diversity in Population
Oligodendrocytes
CNPase3-5%
Fibroblasts
Vimentin55-65%
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Material composition affects cell type diversity
RGD/YIG/NID
More Neurons
*
RGD/YIG/IKV/VSW
More Astrocytes
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Mechanical Stimulation of Neuronal Differentiation
Control of Neuronal Division??
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The Methods
§ Polyacrylamide: Well established material in mechanosensing
Activate coverslips
Pipette gel solution
onto activated coverslips & cover
After solidification
remove top coverslip and treat with Collagen I
Young's modulus vs. cross-linker fraction as determined by mechanical testing(Rowlands, A. S., P. A. George, and J. J. Cooper-White (2008) AJP: Cell Physiology 295.4: C1037-1044)
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Mechanical Influences on Asymmetric division (and ultimately differentiation)
• Extent of C17.2 stem cell differentiation depends on the stiffness of the substrate
• Softer substrates promote differentiation into neurons and the formation of longer neurite extensions, as shown by analysis of the expression of neuron-‐specific β-‐tubulin III
140 Pa 60000 Pa
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Some Surfaces lead to 100% neuronal populations
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Proliferation and Neurogenesis
§ The degree of cell proliferation is controlled in part by the degree of asymmetric cell division
§ When a progenitor cell divides – it can make any variety of cell combinations: ú Two progenitors ú One progenitor and one
neuron ú One progenitor and one
astrocyte ú Etc.
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Differences in Division Type?
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Results
Asymmetric Division during Differentiation on Various Substrates
During differentiation, number of asymmetric divisions is greater on glass over the “soft”
surfaces.
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Results
Cell Death is also reduced on “soft” surfaces compared to glass.
!2#
0#
2#
4#
6#
8#
10#
12#
14#
7.50%# 3.75%# 1.875%# 0.94%#
Num
ber'pe
r'day'
Percent'Serum'
Cell'Death'
140#Pa#gel# Collagen#Coated#glass# Glass#
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Cells grown on “softer surfaces” form functional synapses (red: synaptophysin, green: Homer)
Softer surfaces yield a more homogeneous, more mature population of neurons
Results
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Can division events be controlled?Can division events be correlated to fate?
§ Nanomaterials§ Nanomanipulation
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Nanomaterials
Concentration Dependent Effect
Useful for Imaging, Sensing, Etc.
Long-term Impacts?
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Nanomaterials
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Nanomaterials Hypothesis
NSC
Day 23
Day 10 Asymmetric Division Peaks
Day 5 Cell Division
Peaks
Day 12-‐14 Most prominent morphological
changes Tuj1 expression
peaks
Day 10 Nestin
expression falls
CNTs are interacting with cytoskeleton, altering the division dynamics ** early redistribution of actin (i.e. mechanical remodeling!) CNTs are altering gene expression, but how?
Substrates are forcing cytoskeletal changes, altering the division dynamics, force transduction, and subsequent gene expression
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Nanomaterials Data
0 10000 20000 30000 40000 50000 60000 70000
Regular DNACNT DNA RNACNT RNA
Total Population for day 5 addition
• When materials are added at day 5, the population decreases in the presence of CNT.
• When added at day 10 and day 15, a slight increase is observed.
0
0.1
0.2
0.3
0.4
0.5
Regular DNAc DNA RNAc RNA
Low Concentra,on
• The presence of CNT increases the % of cells expressing β-tubulin III.
• There is an increase in % of cells which do not express phenotypic protein markers tested to date
• ~50% of “Low” express nestin
• Internal destabilization or oompliance changes?
Fraction Neuronal
Low conc
High conc
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Can division events be controlled?Can division events be correlated to fate?
§ Nanomaterials§ Nanomanipulation
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Nanomanipulation
In collaboration with Daniel Ou-Yang, Dimitrios Vavylonis, and Susan Perry
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Nanomanipulation Hypothesis
In collaboration with Daniel Ou-Yang, Dimitrios Vavylonis, and Susan Perry
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Implications? § Establishing a causal link between division mode (and related factors) and final cell fate
§ Control of differentiation = potential therapeutic solution
§ Advanced understanding of division mechanisms
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Much more work to be done…
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Questions?