Dynamic Regulation of the Pluripotent Stem Cell Structure-Function Relationship through Topographical Cues.

Abstract

Pluripotent stem cells (PSCs) are characterised by their ability to self-renew and to differentiate into tissues comprising the three developmental germ layers of the body. This makes them ideal sources of cells for use in regenerative medicine. However, their use for such purposes is hampered for a number of reasons. Both the reprogramming of somatic cells into PSCs and their differentiation for use in research and the clinic are extremely low yielding. This is in part likely to be a result of adaptation to the artificial culture environment, known to force cell structural away from the native state, resulting in altered gene expression. The mechanisms by which PSCs, specifically, integrate cues from their physical microenvironment remain largely unexplored.

This project aimed to improve the culture environment of PSCs for their enhanced differentiation capacity. It was hypothesised that by altering the physical geometry of the culture substrate, the native PSC structure could be maintained, to improve performance upon differentiation. This work utilised the varying topographies provided by three commercial polyHIPE scaffolds (Alvetex®), and the human EC cell line, TERA2.cl.SP12, as a model PSC lineage.

The three scaffolds of differing topography were considered for their ability to maintain pluripotency; a micro-topography resulted in immediate evidence of differentiation. A mixed micro/nano and nano-topography substrate appeared to maintain pluripotency. Due to its ability to allow complete cell removal, the smaller void size scaffold was used to develop a method for long-term PSC culture. This, however, resulted in gradual cell differentiation, suggesting that a mixed-topography was more suited for PSC growth. Alvetex® Strata has been previously shown to be a suitable substrate for the long-term maintenance of PSCs for enhanced differentiation (up to approximately 50 days). Here, we show that the same effect is possible from a single 10-day conditioning step, we term “priming”, with evidence of enhanced differentiation in vitro and in vivo.

The mechanisms of mechanotransduction in PSCs are poorly understood; the culture system developed here allowed for a direct comparison between two-dimensional (2D, conventional) and three-dimensional (3D) culture with substrate geometry as the only variable. The structure of the actin cytoskeleton, as well as that of intermediate filaments and microtubules, was less-developed after 3D culture. This was attributed to a marked decrease in the nucleation of actin, providing a correlative link to the enhanced differentiation possible after 3D priming. Additionally, there were significant differences in the presence of cell-matrix adhesions as well as evidence of reorganisation of the nuclear lamina. This process was successfully applied to the culture of mouse embryonic stem cells, with comparable effect, demonstrating amenability to functionalisation and scale-up development. Overall this system has allowed for the culture of PSCs for enhanced differentiation and allowed for novel insights into how they interact with their microenvironment through mechanotransduction.