Final Report Summary - GENOMICDIFF (The biophysical regulation of genome function and its role in mesenchymal stem cell differentiation) Little is known concerning the mechanisms through which mechanical stimuli are transduced into regulatory signals within the cell. Gene expression can be regulated through alterations in nuclear architecture, modulating genome function. Mechanical load induces both nuclear distortion and alterations in gene expression. One putative transduction mechanism involves changes in gene expression in response to alterations in nuclear architecture as a result of mechanical perturbation of the cell. Additionally, remodelling of the nucleus occurs during stem cell differentiation, altering nuclear stiffness, and potentially influences nuclear architecture mediated mechanoregulation of gene transcription.While much attention has been given to the effects of mechanical stimuli on mesenchymal stem cell (MSC) differentiation, little is known concerning the mechanisms through which mechanical stimuli are transduced into regulatory signals within the cell. This project investigates the potential for the micro-mechanical environment to modulate human MSC differentiation fate via changes in nuclear matrix and chromatin dynamics. The work aims to test the hypothesis that mechanically induced MSC differentiation is driven by changes in chromatin dynamics caused by nuclear distortion.This project involved many stages and employed a wide variety of techniques. This work principally involved two model systems. First, MSCs were seeded within electro-spun nanofibrous scaffolds and subjected to dynamic tensile strain and induced to undergo fibrochondrogenic differentiation in a 3D environment. In the second model system, MSCs were seeded onto fibronectin coated silicone membranes and again subjected to dynamic tensile strain while under induction along osteogenic, adipogenic and chondrogenic lineages in 2D.Nuclear architecture was differentially modulated by biochemical and biophysical cues in a lineage dependent manner. In the basal undifferentiated MSC, strain induced nucleus elongation, increased actin stress fibre formation, nucleus and cytoskeleton reorientation and lamin A/C reorganisation. This lamin A/C reorganisation was also observed with biochemical differentiation along each of the investigated lineages. With differentiation, the response to strain changes and is lineage dependent. The nuclear lamina reorganises, chromatin condensation increases, and histone acetylation associated with gene silencing and heterochromatin formation increases. Lineage also modulates cell and nuclear orientation to applied strain with the nucleus orientation in chondrogenic cells tending to be offset from the cell orientation which may indicate a mechanism through which the cell controls nuclear strain transfer. The mechanisms behind strain-induced chromatin condensation were further investigated and this was found to be ATP-dependent. Chromatin condensation was found to persist for durations corresponding to the length of strain application, and altered the manner in which nuclei deformed in response to stretch. Interestingly, the number of strain episodes increased the magnitude of chromatin condensation and upregulated expression of genes associated with chromatin movement and remodelling. This finding suggests that MSCs may establish a mechanical memory encoded within the nuclear architecture which sensitises the cells to future loading events.Mechanical stimuli play an important role in directing MSC lineage specification. However, the mechanism by which MSC fates are regulated is still poorly understood, and the role of nuclear structure and mechanics in this process has not yet been fully elucidated. Here we show that MSC nuclear architecture is differentially modulated by biochemical and biophysical cues in a lineage dependent manner such that biophysical effects are dependent on the biochemical environment. We additionally show that mechanical stimulation results in a rapid remodelling and condensation of nuclear chromatin, that loading duration influences the persistence of these changes, and that loading establishes a mechanical “memory” in the nucleus, priming cells to respond to further mechanical perturbation.