OUTLINE
Table of acronyms ABSTRACT
1. INTRODUCTION 2. Stem cell differentiation
2.1 Stem cells biochemical neuronal differentiation
2.1.1 Embryonic stem cells biochemical neuronal differentiation 2.1.2 Mesenchymal stem cell biochemical neuronal differentiation 2.1.3 Neuronal stem cell biochemical differentiation
2.2 Micro and nanotopography directs stem cell neuronal differentiation 2.2.1 Topography of the ECM
2.2.2 ESCs maintenance and differentiation on nanopattern 2.2.3 MSCs differentiation on nanopattern
2.2.4 NPs differentiation on nanopattern
2.3 Matrix elasticity directs stem cell neuronal differentiation 3. Cell-substrate interaction
3.1 Cell-substrate adhesion 3.2 Integrins and focal adhesion 3.3 Mechanotransduction:
3.3.1 Mechanotrasmission and mechanosensing 3.3.2 Mechanoresponce
Summary of the state of the art Open questions
4. METHODS
4.1 Emryonic stem cell culture and differentiation 4.1.1 ESCs culture
4.1.2 ESCs neuronal differentiation 4.2 Immunohistochemistry (IHC) 4.2.1 Statistical analysis 4.3 Nanoimprinting lithography 4.3.1 Master fabrication
4.3.2 Fabrication of PDMS nanopillars and grooves 4.3.3 Fabrication of glass pillars
4.4 Submicron imaging:
4.4.1 Scanning Electron Microscopy 4.4.2 Atomic Force Microscopy
4.5 Force spectroscopy measurements:
4.5.1 Force spectroscopy on substrates
4.5.2 Single cell force spectroscopy (SCFS) analysis 4.6 Data analysis:
4.6.1 Immunofluorescence images 4.6.2 Force spectroscopy measurements
4.6.3 Single cell force spectroscopy measurement
5. RESULTS AND DISCUSSION I: PDMS substrates 5.1 PDMS substrates for neuronal differentiation
5.1.1 ESCs direct differentiation on PDMS VS glass 5.1.2 Neuronal precursors survival on PDMS
5.2 Effect of the nanopatterned substrates on neuronal differentiation: Nanopillars Vs Nanogrooves 5.3 Discussion
6. RESULTS AND DISCUSSION II: Role of the nanotopography on the differentiation induction 6.1 Effect of the pillars period and height
6.2 Characterization of neuronal culture
6.3 Quantification of the residual population of primary ESCs 6.4 SEM analysis to study the cell-substrate interaction 6.5 Effect of the geometry on the neuronal differentiation 6.6 Discussion
7. RESULTS AND DISCUSSION III: Role of the mechanical properties on the differentiation induction
7.1 Pillar substrates effective Young’s Modulus
7.2 Neuronal differentiation in relation to pillar substrates effective Young’s Modulus 7.3 Discussion
8. RESULTS AND DISCUSSION IV: Mechanism of neuronal differentiation induction 8.1 Time course analysis of neuronal differentiation
8.2 Cell-substrates adhesion 8.3 Discussion
9. Final discussion and future prospective 10. CONCLUSIONS
11. BIBLIOGRAPHY 12. APPENDIX I 13. APPENDIX II
