title: Directing Neural Stem Cell Differentiation Using Nanopatterned Substrates and Visualization of the Developing Nervous System
creator: Gojak, Christian Philip
subject: 570
subject: 570 Life sciences
description: The development of neural stem cells is regulated by a variety of growth and cell  adhesion factors. A promising approach to direct their differentiation in vitro is the  generation of cell substrates that replicate the intricate physical and biochemical  properties of their cellular environment.   The primary aim of this work was to develop cell substrates that mimic these  properties and explore their potential in directing the differentiation of embryonic  neural stem cells and in supporting axonal outgrowth. For this, gold nanopatterned  glass substrates were used for the controlled immobilization of proteins. The resulting  substrates feature nanopatterned cell signaling proteins at variable surface density, as  well as additional cell adhesive molecules. The role of both factors in regulating the  differentiation of mouse embryonic neural stem cells was investigated independently.   Differentiation of neural stem cells on substrates uniformly coated with the cell  adhesion molecules laminin, fibronectin and N-cadherin showed no effect on the in  vitro generation of newborn neurons in comparison to polyornithine controls.  Moreover, a 2,5-fold increase in cell number was observed on all cell-adhesion  molecules, indicating an increase in cell proliferation. Furhermore, the role of cell  adhesion molecules in supporting axonal outgrowth of dorsal root ganglia explants  was investigated. The longest axonal projections ranging up to 800µm could be  observed on laminin-coated substrates. In contrast, outgrowth on fibronectin as well  as fibronectin-derived peptide nanopatterned substrates resulted in 200-250µm long  projections.  The role of nanopatterned Notch cell receptor ligand Delta-like 1 (Dll-1) substrates in  directing the differentiation of neural stem cell cultures was investigated using  variable ligand densities. As a result, Notch activation resulted in increased neurite  number, branching, and cell body size. The strongest response was observed using  340 ligands/µm2 (56nm interparticle spacing) in comparison to 735 ligand/µm2  (90nm) and uniformly coated Dll-1 substrates. Additionally, a small fraction of drastically enlarged neurons was observed on Dll-1 substrates, but no effect on the  total number of newborn neurons.   The next aim of this thesis was to develop a system for the non-invasive visualization  of the embryonic nervous system. For this, a transgenic mouse line that expresses  high levels of green fluorescent protein (GFP) specifically in newborn neurons was  characterized. Imaging using light sheet fluorescence microscopy resulted in high-  resolution visualization of the entire nervous system in whole embryos allowing for  generation of three-dimensional models and virtual specimen sectioning. Additionally,  this technique was successfully applied for the visualization of innervation defects  caused by mutation of the axonal-guidance protein Semaphorin 3A.  In summary, the nanopatterned protein substrates presented in this work constitute a  powerful tool for influencing in vitro development of neural stem cells. Additionally,  the introduced transgenic mouse featuring GFP-expressing neurons allows for highly  detailed visualization of the developing nervous system in whole embryos. 
date: 2012
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/13451/1/CGojak_Diss_2012_alt.pdf
identifier: DOI:10.11588/heidok.00013451
identifier: urn:nbn:de:bsz:16-opus-134511
identifier:   Gojak, Christian Philip  (2012) Directing Neural Stem Cell Differentiation Using Nanopatterned Substrates and Visualization of the Developing Nervous System.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/13451/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng