Examination of human mesenchymal stem cell behavior in the presence of individual whole extracellular matrix protein in three-dimensional environments.

摘要

Stem cell behavior is guided by the chemical, mechanical, topographical and biochemical properties that are provided by the extracellular microenvironment. While a number of experiments have showcased the strong influence biochemical signals from extracellular matrix (ECM) proteins can have on stem cell behavior, it has been difficult to elucidate the true impact of individual ECM proteins in three-dimensional environments. In this work we utilized multiphoton excited photochemistry to generate 3D, whole ECM protein structures containing collagen I, fibronectin, and laminin.;Human mesenchymal stem cells were cultured on these 3D structures and RNA sequencing was performed to broadly examine how gene expression, an indication of cell behavior, was altered by the presence of individual whole ECM proteins. We noted that stem cells cultured in 3D environments with individual ECM proteins were capable of remodeling the external environment both physically and biochemically. Additionally, these modifications depended on the duration of culture, whether the cells were exposed to 2D or 3D environments, and the presence of specific ECM proteins. A broad examination of the RNA sequencing data revealed differences in gene expression profiles of stem cells between 2D standard culture and 3D structure, and among different 3D structure types. Further examination of the data revealed that long-term culture of embryonic stem-derived mesenchymal stem cells in media lacking chemical induction components in 2D standard tissue culture and 3D environments primarily impacted osteogenic differentiation.;Additionally, we examined the expression levels of genes associated with endothelial and smooth muscle cells, two cells vital to vasculature development, in structures containing fibronectin. We observed the presence of genes for endothelial and smooth muscle cells, indicating that fibronectin may influence vasculature development through the differentiation of these two cell types.;Additionally, we noted a decrease in sphingosine 1 phosphate lyase 1 in fibronectin structures, which may effect the degradation of sphingosine-1-phosphate, an important factor in vasculature development. In conclusion, we utilized multiphoton excited photochemistry-based 3D printing and RNA sequencing to examine how the presence of individual ECM proteins in 3D direct stem cell behavior. And the combination of these two technologies enabled the exploration of ECM-stem cell dynamics and may facilitate approaches to control stem cell response for clinical applications.