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Biophysical Manipulation of Adult Stem Cell Epigenetics (BioMASCE) By Cell-Penetrating Nanoneedle Substrates

Periodic Reporting for period 1 - BioMASCE (Biophysical Manipulation of Adult Stem Cell Epigenetics (BioMASCE) By Cell-Penetrating Nanoneedle Substrates)

Reporting period: 2016-06-01 to 2018-05-31

Signals from the extracellular environment are highly influential on cell behaviour. In particular, physicochemical cues from the materials upon which cells are cultured can drive fundamental changes to cell identity. Over the past two decades, a number of studies have demonstrated that complex interactions between cells and engineered materials can be manipulated in order to generate desired outcomes in cell fate and function, but these have consistently targeted the cell membrane and therefore have indirect consequences on intracellular machinery. In contrast, materials that exhibit the ability to simultaneously influence the classical machinery located at the cell membrane while also modulating the functions of intracellular processes, such as gene transcription or organization of intranuclear cargo, would provide new bioengineering methods for controlled regulation of cell function.
In this project, porous silicon nanoneedle (nN) arrays were interfaced with human mesenchymal stem cells (hMSCs) in order to directly affect multiple components of the cell. Our findings indicate that nN interfacing leads to a range of biophysical changes within the cell, including accumulation of structural components of the nuclear envelope, functional decoupling of mechano-sensitive transcriptional regulators, and disruption of a steady-state epigenetic program within the cells. This work has showcased the ability of nano-structured materials to regulate high-level biological functions in human stem cells, and offers unique insight for applying next-generation materials for directing cell behaviours in the context of tissue engineering, regenerative medicine, and stem cell biology.
The following work was performed during this fellowship:
- nN interfacing disrupted several mechano-responsive cellular elements. Overall changes to cell and nuclear morphology were characterized/quantified using high-content image analysis and computer-assisted feature extraction algorithms. From these data, actin bundling emerged as a dominant feature modulated during culture on nN substrates, which indicated that cellular biomechanics were heavily regulated in this system. Furthermore, we demonstrated that portions of the classical mechano-responsive machinery were not performing as would be expected when cultured on a stiff material such as porous silicon.

- In order to evaluate the functional consequence of these events, the activity of a mechano-responsive co-factor was measured. Surprisingly, culture on nN resulted in cytosolic localization of the factor, as well as inactivation of its functionality. Therefore, this system allows for the decoupling of various biomechanical factors that have been exceptionally challenging to study in the literature – namely, the relationship between cell spreading, adhesion formation, transcription co-factor localization/activation, and substrate mechanics – thereby providing new insight into how cells sense and respond to (nano)material-derived cues.

- Focusing on the nucleus, nN interfacing clearly displaced the nuclear envelope and intranuclear cargo, as evidenced through super-resolution imaging. When evaluated further, the localization of lamins (the structural proteins of the nuclear envelope) were shown to diverge. These data indicate that the nucleus dynamically responds to physical perturbations caused by nN interfacing, which has not been shown previously in the literature. Furthermore, analysis of epigenetic changes, including histone modification patterns and expression of histone-modifying proteins, indicated that nN interfacing directly alters the epigenome of hMSCs.

- Secondment to The Technology Partnership (TTP): Over the course of a three-month period, the fellow spent 2-3 days / week seconding at TTP, a technology innovation consultancy located in Cambridgeshire, United Kingdom. During this secondment, the ability of nN to act in concert with proprietary technologies from TTP were evaluated. The results from this secondment suggest that these combined methods show great promise for clinically-relevant, commercializable techniques that will continued to be developed during an ongoing collaboration between TTP and the host laboratory.

- Outreach Activities – During the two year funding period, extensive outreach was performed to engage with young students and encourage them toward a career in science. For example, in 2016 the fellow gave a one-hour lecture for the London International Youth Science Forum, held at Imperial College London, which was followed by several hands-on demonstrations by colleagues in the laboratory.

Final Result Overview
The data generated in this project indicate for the first time that one piece of the mechano-responsive pathway (focal adhesions) is a critical regulator for determining the activity of the co-factor YAP. Because of the ability of our nanoneedle-based system to effectively decouple cell parameters that are intrinsically linked (such as cell area, focal adhesion formation, and actomyosin activity), these data provide information that has been thus far impossible to elucidate using other material systems. This work has been presented at three international conferences (TERMIS Boston 2015; TERMIS-EU Stockholm 2016; NanoGE Berlin 2016) and has been prepared as a high-level manuscript that is currently under revision.
A range of techniques were employed to complete this project, requiring extensive interdisciplinary collaboration and insight. For the preparation of nN substrates, microfabrication and photolithography were used, as well as surface functionalization methods. For cell-based experiments, classical molecular biology techniques such as quantitative polymerase chain reaction (qPCR) and confocal microscopy / immunocytochemistry were used, but advanced methods such as three-dimensional structured illumination microscopy (3D SIM, super-resolution microscopy) were critical to develop the insights that eventually became the highlights of this work. Finally, high-content image analysis was performed to extract cell morphology features and protein localization patterns, which were invaluable pieces of data to support our research; these types of techniques are rarely combined to such an advanced degree, which provided a level of completeness that for our analysis and subsequent understanding of the cellular processes.
The outcomes from this project will deliver a significant impact within the scientific community due to the advancement of knowledge within the fields of mechano-biology and cell/nano-material interactions. However, by progressing these fields, it is possible to enhance the general understanding of how cells sense and respond to external stimuli in the context of human health and disease. Mechano-biology is a topic of great interest at the moment because it has become increasingly clear that the mechanical properties of cells and tissues play a role in progressing diseases such as cancer and atherosclerosis. Therefore, in using the nN system to uncover new details for the roles of different players in the mechano-biology pathway, the findings from this project contribute to the ongoing literature and create a more complete picture of this complex process. The data collected during this project will undoubtedly be used to further clarify our understanding of cell mechano-biology in the context of clinical pathologies.