The Extracellular Environment's Effect on Cellular Processes: An In Vitro Study of Mechanical and Chemical Cues on Human Mesenchymal Stem Cells and C17.2 Neural Stem Cells

Stem cells are widely used in the area of tissue engineering. The ability of cells to interact with materials on the nano- and micro- level is important in the success of the biomaterial. It is well-known that cells respond to their micro- and nano-environments through a process termed chemo-mechanotransduction. It is important to establish standard protocols for cellular experiments, as chemical modifications to maintenance environments can alter long-term research results. In this work, the effects of different media compositions on human mesenchymal stem cells (hMSCs) throughout normal in vitro maintenance are investigated. Changes in RNA regulation, protein expression and proliferation are studied via quantitative polymerase chain reaction (qPCR), immunocytochemistry (ICC) and cell counts, respectively. Morphological differences are also observed throughout the experiment. Results of this study illustrate the dynamic response of hMSC maintenance to differences in growth medium and passage number. These experiments highlight the effect growth medium has on in vitro experiments and the need of consistent protocols in hMSC research. A substantial opportunity exists in neuronal research to develop a material platform that allows for both the proliferation and differentiation of stem cells into neurons and the ability to quantify the secretome of neuronal cells. Anodic aluminum oxide (AAO) membranes are fabricated in a two-step anodization procedure where voltage is varied to control the pore size and morphology of the membranes. C17.2 neural stem cells are differentiated on the membranes via serum-withdrawal. Cellular growth is characterized by scanning electron microscopy (SEM), ICC and qPCR. ImageJ software is used to obtain phenotypic cell counts and neurite outgrowth lengths. Results indicate a highly tunable correlation between AAO nanopore sizes and differentiated cell populations. By selecting AAO membranes with specific pore size ranges, control of neuronal network density and neurite outgrowth length is achievable. To understand differentiation marker expressions in C17.2 NSCs and how material stiffness affects differentiation, cells are cultured on substrates of varying stiffness. qPCR is used to analyze neural stem cell, neural progenitor cell, neuron-restricted progenitor and differentiated post-mitotic neuronal cell RNA expression. Results suggest a relationship between material stiffness and neuronal development in C17.2 neural stem cells.