title: Living biointerfaces to direct stem cell fate
Creators: name: Petaroudi, Michaela
Publisher: name: University of Glasgow
Year: 2022

description: Cellular therapeutics is a constantly evolving field within tissue engineering and regenerative medicine, that aims to provide clinically relevant solutions for tissue repair applications. Currently, the most commonly used methods of tissue regeneration revolve around the use of stem cells, due to their inherent potential to self-renew and differentiate, driving damaged tissue repair. However, the scarcity of suitable donors, the variety of challenges associated with primary stem cell isolation, the lack of controllable ways to direct differentiation into specific lineages, and insufficient numbers of engraftable cells, have hindered the extensive and efficient use of stem cells in clinical settings. These issues, that stem from the lack of understanding of the underlying mechanisms driving stem cell differentiation, have created the need for the study of stem cell behaviour and the chemical and mechanical stimuli that influence their fate decisions. In an attempt to understand the specific mechanisms that drive stem cell survival, proliferation and differentiation, research efforts have been focused on the engineering of physiologically relevant cell microenvironments, that could mimic the natural stem cell niche. The endeavour to provide close niche analogues has driven the evolution of such efforts from initially simple cell culture methods, based on the use of stem cell expansion media containing soluble growth factors, to the development of dynamic surfaces, bioactive 3D substrates and bioprinted scaffolds with the aim to provide close representations of the target cell niche. Given their enormous clinical potential to treat a variety of hematological disorders and heal major tissue injuries respectively, Hematopoietic (HSCs) and Mesenchymal stem cells (MSCs) have been the two most widely used cell types in cellular therapeutics. Currently, most common scientific efforts for the expansion of HSCs have been focused on cell cultures with the external addition of niche growth factors including stem cell factor, thrombopoietin and FMS-like tyrosine kinase 3 ligand (FLT3L). In contrast, research on clinical applications of MSCs has focused on identifying the soluble cues that drive cell differentiation into specific lineages, such as bone repair. However, despite the variety of scientific studies aiming to increase the therapeutic uses of both stem cell types, progress is still slow in the development of efficient ways to produce clinically relevant cell numbers to address the increasing need for cellular grafts. Active biomaterials have recently received increased scientific attention due to their intrinsic ability to exert instructive or stimulating effects on cells and tissues by engineering the material’s responsiveness to internal or external stimuli that can promote tissue repair and regeneration. In particular, living interfaces have the potential to actively produce and deliver growth factors of interest to the cultured cells and guide their behaviour to improve tissue regeneration, providing a promising opportunity to substantially enhance the efficacy of tissue engineering and regenerative medicine. In this work, we have genetically engineered the non-pathogenic bacterial species Lactococcus lactis (L. lactis) to produce recombinant human CXCL12, thrombopoietin, and VCAM1 and have combined these populations with the previously developed FN-expressing L. lactis to create bone marrow niches ex vivo. The purpose of this work is to engineer a platform that could directly influence stem cell fate by actively stimulating the cultured cells by the recombinant proteins and by added 3D elements, such as poly(ethylene) glycol hydrogels. The successful development of such a system could have significant potential in cellular therapeutics, as it could provide a variety of physiologically relevant, niche mimicking stimuli to encourage stem cell survival and proliferation. In particular, our platform could be used to maintain HSCs and MSCs in a naïve state, while also encouraging their self-renewal and proliferation. In alignment with the increasing demand for HSC and MSC transplants for clinical applications, our work could provide insights into the most optimal culture methods that would encourage the proliferation of the cultured stem cells, in order to produce clinically relevant cell numbers for the cellular therapeutics. In parallel, our system could be used as a platform to study a variety of aspects of the bone marrow, such as the effects of specific soluble and mechanical stimuli on stem cell fate in healthy and deregulated conditions. The results of this thesis suggest that L. lactis can be used as a versatile tool to produce a variety of recombinant proteins. Its ability to form stable biofilms enables the bacteria to act as a living interface between the substrate below and the stem cells seeded above. We report that HSCs cultured on top of L. lactis biofilms show notable expansion, and decreased tendency to differentiate, both in 2D where the HSCs are seeded directly above the biofilms, and in 3D where the stem cells are maintained in a hydrogel, on top of the biofilms. In parallel, we report that MSCs cultured on our living interfaces display maintenance of their naïve, stem-like phenotype, without showing commitment to differentiated cell lineages. In the future, this tuneable, biocompatible system can be engineered to produce any recombinant protein or small molecule and deliver it to any cultured cell type. These expressed factors can be either secreted or presented as a membrane protein on the bacteria, providing opportunities for both soluble and mechanical stimulation of the cultured cells. The variety of combinations of recombinantly expressed factors and cultured cell types provide the opportunity for the development of different niche-mimicking microenvironments that can be tailored to address different clinical needs. In total, our active biointerface provides a proof of concept that living materials can be successfully engineered and used in biomedical applications.