The ultimate goal of regenerative medicine is to create functional engineered biomaterial to repair or replace the damaged tissues. My work firstly was to perform multi-scale characterization on the collagen hydrogels used as scaffolds. Different initial collagen concentrations and incubation temperatures were identified to affect the properties of collagen hydrogels, such as the microstructure obtained with second harmonic generation imaging (SHG), nanostructure imaged with transmission electron microscopy (TEM) and molecular structures detected with Raman spectroscopy. Comparing to the real tissues, the drawback of collagen hydrogels is the lack of mechanical strength. Cross-linking has been known to increase the mechanical strength. I then characterized the effect of different cross-linking reagents including glycation reagents, genipin and 1-Ethyl-3 (3dimethylaminopropyl) carbodiimide (EDC) on the collagen hydrogel. SHG imaging showed that glycation reagents and genipin modify the microstructure within collagen hydrogel but EDC has no effect on the microstructure. TEM images revealed that glycation reagents and genipin also remove the native-like striation pattern in nanostructure but EDC has no effect on it. Raman spectroscopy showed that glycation reagents and EDC affect the molecular information of collagen hydrogels. Lastly, two different types of stem cell, mouse embryonic stem cell (ES cell) and induced pluripotent stem cells (iPS cells) were seeded to the collagen hydrogels and then examined the effect of different structure of collagen hydrogel on the neuronal differentiation of stem cells. The behaviors of G-Olig2 ES cell and a7wt iPS cell were compared during early differentiation with retinoic acid into a neural lineage employing separately the encapsulated or topographic 3D collagen hydrogel models. For the encapsulated model, the differentiation of ES cell and iPS cell were slower for the cells encapsulated within 4 g/l collagen hydrogels compared to the cells in 2 g/l hydrogels. The differentiation process of both cells types seeded on top of the 3D collagen hydrogels was affected by the structure modified by cross-linking. For example, the differentiation rate of ES cells was slower on the cross-linked hydrogels but the differentiation rate of iPS cells was faster on the cross-linked hydrogels.