IBEC PhD Discussions Sessions: Gizem Altay and Marta Pozuelo

Towards bioengineering a platform for the growth and differentiation of intestinal stem cells

Intestinal diseases affect major part of the world population; yet, there is still the need of developing effective treatments. Animal models are widely used to study these diseases; however, many intestinal processes are difficult to control in vivo. Besides these models are very costly and they raise ethical concerns. In vitro models, on the other hand, can enable improved studies in a well-controlled and ethical manner. The most advanced in vitro model reported up to date is the organoid system. Organoids are stem cell derived 3D self-organized tissue models that recapitulate many biological parameters of the native tissue such as the spatial organization, multicellular hierarchy, cell-matrix interactions and some physiological functions. Intestinal organoids contain crypt-villi domains with all major intestinal cell types and a central lumen region, resembling many aspects of the intestine in vivo. However, it is often difficult to control the stem cell differentiation and cell-matrix interactions within these systems. Besides, the luminal compartment being inaccessible is a major inconvenience for studies such as drug absorption. These drawbacks raise the need to bioengineer a platform to obtain an intestinal epithelial model from intestinal stem cells.

In the intestinal epithelium the stem cell growth and differentiation are controlled by biochemical gradients of the stemness factors. Moreover, the 3D architecture and the mechanical properties of the native tissue are influential in the phenotype of these cells. Therefore, in bioengineering the cellular microenvironment, it is very important to reproduce the structural and mechanical characteristics of the native intestinal epithelium and to provide cells with the appropriate biochemical cues.
In this study we have developed 3D villus anatomical models from extracellular matrix like soft polyethylene glycol based hydrogels. The platform developed has controlled biochemical functionality and supports the intestinal stem cell attachment and growth. It is characterized to be able to create spatio-chemical gradients of the stemness factors to further modulate stem cell growth and differentiation. We have also developed a way to obtain 2D epithelial monolayers from intestinal crypts to further be implemented in the hydrogel based 3D villus models.

Studying charge transport in Single-Protein wires

Electron Transfer (ET) is undoubtedly one of the most important processes in life. Molecularly well-defined ET pathways in complex protein ensembles play a vital role in biological processes such as cell respiration or photosynthesis. The fundamental understanding of ET processes in biology is important not only to understand such key natural processes but also to advance in the design of biomolecule/electrode interfaces for Bioelectronic applications. The development of new techniques such as scanning probe microscopies (SPM) played a key role. In particular, the electrochemical scanning tunneling microscopy (ECSTM) has been exploited to in situ monitor the ET rate as a function of the applied potential of individual metalloproteins immobilized on an Au electrode thanks to the single-molecule spatial resolution and the electrochemical gate capabilities.

Azurin from Pseudomonas aeruginosa is a widely studied redox protein model both in bulk and at the single molecule level. Its globular structure contains a coordinated copper ion, which makes the protein capable of exchanging electrons by switching its redox state (Cu I/II) and supports its role as a soluble electron carrier in the respiratory chain of bacteria.

In this contribution, we will show our advances on the design and characterization of single-protein devices using a Cu-Azurin metalloprotein model. We have demonstrated transistor like-behaviour in an electrochemically-gated single-protein wire that operates at very low voltages thanks to the Cu-Azurin redox properties. It was demonstrated that the conductance varies depending on the redox state of the Cu centre, having its maximum value at the redox-midpoint. We have also analysed the spontaneous formation of single-Azurin electrical contacts through the monitored current when the two ECSTM electrodes were placed at a fixed distance. Discrete switching events for the conductance were observed, whose frequency depends on the applied electrochemical conditions and, therefore, they were univocally ascribed to discrete changes in the redox state of the trapped protein.

In order to tailor the charge transport behaviour of the single-protein wire, we have synthesized several mutants of the protein by exploiting point-site bioengineering schemes at different positions of the protein second coordination sphere. Our results show that we can rationally change the transport mechanism of the single-protein device by studying the effect of the specific residue modification on the particular ET pathways in the protein backbone.

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