Generally, in this thesis, we studied the effect of diverse mechanical environments including different extracellular matrix stiffness and varied hydrogel elasticity on the behaviors and functions of cancer cells and stem cells. Specifically, two topics are included in this thesis. In the first topic, the impacts of 2D substrate stiffness on the cancer stem cell (CSC) maintenance, chemotherapeutic response and autophagy activation in breast cancer cells was investigated. While in the second topic, we focused on the impact of the 3D environment and hydrogel elasticity on the proliferation and self-renewal ability of mouse iPS cells. Taken together, no matter culturing on 2D substrates or encapsulating in 3D hydrogels, the mechanical environment provided by extracellular matrix indeed makes a great influence on the phenotypes and gene expressions in either cancer cells or stem cells. In the first project, to study the impact of extracellular matrices stiffness on breast cancer cell functions, we used FN coated polyacrylamide hydrogels as substrates to culture breast cancer cells. By adjusting the ratio and concentration of acrylamide and bis-acrylamide, we fabricated three different elastic substrates with elasticity of ~0.48 kPa, ~4.47 kPa, ~34.88 kPa, which I named “soft” ”median” ”stiff” substrate. First, we showed the diversity of cellular morphology of MCF-7 cells on different substrates, which is consistent with the previous studies. Cells spread widely on stiff substrate while barely spread on soft one, which may be the main cause of increasing proliferating capacity along with the increased stiffness. Next, we found the different chemotherapeutic response of breast cancer cells on different substrates. Breast cancer cells showed enhanced chemosensitivity to doxorubicin and cisplatin but not cyclophosphamide when matrix stiffness increased. To figure out the main reason for matrix stiffness-dependent different chemotherapy response, we detected the stemness characteristics of breast cancer cells on different substrates by analyzing the changes in the cancer stem cell (CSC) population. And we found that the CSC population became smaller and smaller when matrix stiffness increased. On the other hand, cells cultured on stiff substrate generated less tumorspheres in the CSC enrichment experiments. These data indicated that the soft substrate could maintain the CSC population in breast cancer cells. Another focus of this project is autophagy. By creating an environment of nutrient deprivation, we found that the level of activated autophagy was highest on stiff substrate and lowest on soft one, in other words, along with the increasing matrix stiffness, autophagy increased. And interestingly, the increased autophagy was suppressed when actin cytoskeleton and stress fiber was disturbed by adding F-actin inhibitor or non-muscle myosin inhibitor. These results demonstrated integrated actin cytoskeleton tension is required for autophagy activation. We also involved YAP in the matrices stiffness-mediated autophagy regulation. The knockdown of YAP greatly reduced the autophagy levels in all group, however, the increasing trend of autophagy along with increasing stiffness remained unexpectedly. This indicated that the regulation of autophagy by matrix stiffness is independent of YAP. To further confirm this discovery, we next detected YAP nuclear translocation on different substrates and found that in normal breast cancer cells, almost all the YAP located in the nucleus, in other words, YAP nuclear translocation was not influenced by stiffness. In contrast, in breast cancer stem cells (BCSC) YAP nuclear translocation was inhibited on the soft environment which indicated that YAP is a crucial factor in the regulation of the BCSC population. This concept was also proved by the result that the knockdown of YAP eliminated the difference of CSC population among different substrates. Last but not the least, the increasing trend of autophagy induced by increasing stiffness could be eliminated by Rho inhibitor, ROCK inhibitor, and ERK inhibitor, indicated that Rho-ROCK-ERK signal pathway could be involved in the regulation autophagy by matrix stiffness. But more evidence is needed to strengthen this viewpoint in the future. In addition, the relevance between chemotherapy sensitivity and autophagy activation on different substrates is also worth exploring, which was missing from this project. In the second project, a chemical defined hydrogel based on polyethylene glycol (PEG) and dendritic polyglycerols (PGs) was manufactured to build a more physiological 3D environment. Utilizing microfluidic chip, mouse iPS cells were encapsulated in microgels of 200 μm in diameter. Multicellular spheroids also called embryoid bodies were formed inside the microgels which were floating in the culture medium. Comparing with traditional suspension culture, more but smaller embryoid bodies with high proliferative capacity were generated within the microgels which lead to a better expansion curve. Further detection showed that microgel encapsulated iPS cells had equivalent or better pluripotency compared with that in traditional suspension culture. By increasing the concentration of polymers, more stiff hydrogels with low elasticity were generated but accompanied by rapidly decreased cell viability and proliferative ability as well as the amount of embryoid body. In this project, we wanted to explore the effect of hydrogel elasticity on the proliferative capacity and self-renewal ability of iPS cells. However, the extremely low cell survival rate made this impossible. In the future, more work is needed to generate hydrogels with proper elasticity.