Improving Fibroblast to Neuron Reprogramming through Engineering of the Cell Microenvironment
- Author(s): Kelkhoff, Douglas Oscar
- Advisor(s): Li, Song
- et al.
Cells respond to environmental cues to inform expression. This function is quintessential for effective tissue development and coincides with chemical signaling to depict a host of signals that predispose progenitor cells to adopt a lineage commitment and differentiated cells to adopt mechanoregulated phenotypes. Herein the mechanotransductive effects of substrate stiffness and ligand presentation are explored as they pertain to fibroblast to neuron reprogramming using the Brn2, Ascl1 and Myt1l (BAM) viral system and cross referenced against previous studies in the effect of stiffness on mesenchymal stem cells (MSCs). It was discovered that ligand surface density deviations of a single order of magnitude are not sufficient to induce differential reprogramming efficiency. By contrast, stiffness has a profound effect at the intersection of broadly mechanotransductive pathways and neurogenerative genes. Using population RNAseq analysis, it was found that ERK and GSK-3β are largely regulated by viral expression of the singular factor, Ascl1. ERK, in conjunction with SMURF2, combine to have a biphasic regulatory effect on both arms of the SMAD pathway. Furthermore, modern analytical ontology toolsets were used to expose trends in MSC behavior governed by stiffness and cross validation of stiffness dependencies in the induced neuronal system. Similar to reprogrammed fibroblasts, MSCs show a stiffness dependent SMURF2 regulation. Concurrently, surface MHC class was seen to vary across stiffness and the antithrombogenic capacity of MSCs was shown to be stiffness dependent. Collectively this work sets the framework for further exploration into the mechanical regulation which drives progenitor lineage commitment and reprogramming capacity through a common mechanism.