Interactions  of  Neurons  and  Materials  

Sabrina  Jedlicka  

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.  

Primary  Goals  

§  Analyze  the  function  of  the  nervous  system  §  Develop  methods  to  restore  damaged  neurological  functions  

§  Create  artificial  neuronal  systems  

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.  

Cell-­‐Material  Interactions Neural Stem

Cells

Surface-Induced Chemotransduction

Cellular Mechanotransduction

Cellular Biomechanics

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)

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

Research History

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)

Developing Bio-inspired Materials…

Ingber,  D.  E.  (2006).  Cellular  mechanotransduction:  Putting  all  the  pieces  together  again.  Faseb  Journal,  20(7),  811-­‐827  

C17.2 Neural Stem Cells

§  Neuronal differentiation is controlled via serum withdrawal (starvation), which usually results in a homogeneous population of neurons

But…

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.    

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!    

NSCs and Mechanosensing: The Research QuestionHow do material cues impact differentiation of NSCs?

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

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

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%

Material composition affects cell type diversity

RGD/YIG/NID

More Neurons

*

RGD/YIG/IKV/VSW

More Astrocytes

Mechanical  Stimulation  of  Neuronal  Differentiation  

Control  of  Neuronal  Division??  

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)

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  

Some  Surfaces  lead  to  100%  neuronal  populations  

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.  

Differences  in  Division  Type?  

Results

Asymmetric Division during Differentiation on Various Substrates

During differentiation, number of asymmetric divisions is greater on glass over the “soft”

surfaces.

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#

Cells grown on “softer surfaces” form functional synapses (red: synaptophysin, green: Homer)

Softer surfaces yield a more homogeneous, more mature population of neurons

Results

Can division events be controlled?Can division events be correlated to fate?

§  Nanomaterials§  Nanomanipulation

Nanomaterials

Concentration Dependent Effect

Useful for Imaging, Sensing, Etc.

Long-term Impacts?

Nanomaterials

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    

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

Can division events be controlled?Can division events be correlated to fate?

§  Nanomaterials§  Nanomanipulation

Nanomanipulation

In collaboration with Daniel Ou-Yang, Dimitrios Vavylonis, and Susan Perry

Nanomanipulation  Hypothesis  

In collaboration with Daniel Ou-Yang, Dimitrios Vavylonis, and Susan Perry

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

Much  more  work  to  be  done…  

Questions?