Utilising non-pathogenic bacteria as a substrate for mesenchymal stem cell adhesion and differentiation

Tissue engineering and regenerative medicine is a constantly evolving field of science that directs the use of cells to repair damaged or diseased tissue. Currently, stem cells are the most widely used source of cells due to their inherent characteristics of self-renewal and differentiation. However, stem cell regenerative therapies are still lacking, upon removal from the body, stem cell grow dysplastically towards unwanted lineages, compounded by their limited number makes the therapeutic potential of these cells difficult to obtain. These problems are due to the lack of knowledge of the underlying systems and mechanisms of phenotypical commitment through differentiation. In an attempt to circumvent these problems, scientists have begun the construction of dynamic surfaces, that is, surfaces that mimic the constantly evolving and changing environment in which stem cells reside in the body, known as the niche. These biomimetic strategies aim to reproduce the physical architecture, chemical composition and plasticity of the in vivo environment in vitro. This physical architecture can provide the cells with behavioural cues, mainly through transmembrane receptors known as integrins which link the extracellular matrix to the cytoskeleton; and therefore convey physical architecture of the environment to the cell. Scientists consciously design biomimetic systems to incorporate integrin binding ligands such as the adhesive tripeptide arginine-glycine-aspartic acid (RGD). The chemical composition of the niche depends on the in vivo milieu, and the needs of the body at this specified time. More specifically, mesenchymal stem cells reside in a variety of niches, with the bone marrow being a prime example. Upon osteogenesis, many chemical signals are delivered to this niche, with arguably the strongest osteogenic signal from bone morphogenetic protein 2 (BMP-2). This growth factor can activate osteogenic genes within the mesenchymal stem cell and predetermine differentiation of the cell towards an osteoblastic fate. In this work, we have developed a genetically engineered non-pathogenic bacteria, Lactococcus lactis to display the III7-10 fragment of fibronectin to allow mammalian cell integrin adhesion. This fragment contains two important sequences, the RGD adhesive tripeptide and the synergy sequence proline-histidine-serine-arginine-asparagine (PHSRN). RGD allows the mammalian cells to interact with the bacteria through promiscuous integrin attachment. The PHSRN sequence binds synergistically with RGD to some integrins, such as α5β1. These bacteria have also been modified to express the osteogenic growth factor, BMP-2 to direct mesenchymal stem cell differentiation towards an osteoblastic fate. These bacteria readily form spontaneous two-dimensional biofilms on a variety of surfaces, and can therefore act as a living interface between the synthetic surface below and the mammalian cells seeded above. The results of this thesis demonstrate that Lactococcus lactis can be used as a successful dynamic surface to control the adhesion, proliferation and differentiation of mesenchymal stem cells. Mesenchymal stem cells seeded over BMP-2 secreting Lactococcus lactis demonstrate decreased cell proliferation at short time points and increased osteoblastic markers at longer time points. Further to this, the interface has been made dynamic by making the bacteria inducible, that is, BMP-2 can be expressed in a temporal manner, and at different concentrations to finely tailor specific protein production. In the future, this system can be further exploited to express or deliver almost any protein or small molecule that can aid in the development of new tissues from their progenitor cells. As demonstrated, these proteins can both be secreted into the medium or displayed as cell wall bound proteins; and can also be constitutively or inducibly expressed. This interface, based on non-pathogenic bacteria establishes a new paradigm in surface functionalisation for regenerative medicine applications.