Human mesenchymal stem cells (hMSCs) are colony-forming unit fibroblasts (CFU-F) derived from adult bone marrow and have significant potential for many cell-based tissue-engineering applications. Their therapeutic potential, however, is restricted by the diminishing plasticity as they are expanded in culture. In this study, we investigated using N-isopropylacrylamide (NIPAM)-based thermoresponsive polyelectrolyte multilayer films as a culture substrate to support hMSC expansion without compromising their stem cell characteristics. The thermoresponsive surface is hydrophobic at culture temperature (37 °C) and hydrophilic at lower temperature (<4 °C), which enabling cell detachment without enzymatic treatment. The thermal responsive PEMU films were made via layer by layer adsorption of thermoresponsive polymers copolymerized with oppositely charged polyelectrolytes. The coating has been made to both 2-D and 3-D constructs. Initial study found that NIPAM-based surfaces supported efficient cell detachment but at lower growth rate compared to the control surfaces. To improve cell adhesion and growth, NIPAM surfaces were further modified by positively charged Poly(allylamine hydrochloride),PAH and negatively charged Poly(styrene sulfonic acid), PSS and compared to FBS coated surfaces. Surface charges were shown to alter the ECM structure and subsequently regulate hMSC responses including integrin expression, adhesion, proliferation, detachment, and colony forming ability. The positively charged thermal responsive surfaces improved cell adhesion and growth in a range comparable to control surfaces while maintaining significantly higher CFU-F forming ability. Immunostaining and Western blot results indicate that the improved cell adhesion and growth on the positive charge surfaces are resulted from the elevated adhesion of fibronectin on the positively charge surfaces. These results demonstrate that layer-by-layer approach is efficient to form PNIPAM-based thermal responsive surfaces for hMSC growth and removal without enzymatic treatment. The results also show that surface charge regulates ECM adhesion, which in turn influences not only cell adhesion but also CFU-forming ability and their multi-lineage differentiation potential. We have also investigated the effects of NIPAM-based coating of 3-D PET scaffold surfaces on cell growth, thermal removal, and multilineage differentiation potential. We will compare the effects of surface properties on hMSC expansion in 3-D scaffold and determine the condition for efficient cell removal from 3-D scaffold while preserving hMSC property. Overall, NIPAM-based surfaces provide many advantages over the conventional culture substrate for hMSC expansion.