Single cell live imaging in vivo, to understand cell activity in the context of regenerative medicine and cancer biology.

Stem cell-based, and more general cell therapies have shown promising results across many disciplines in medicine. However, several recent clinical studies have led to disappointing results, due to the fact that the fate of the transplanted cells is unknown and the fundamental mechanisms underpinning their effects not understood. This project will monitor and track the interaction over time of multiple cell types transplanted in vivo and how they interact with the host tissues. Non-invasive cell imaging techniques are essential to obtain real-time, quantitative, and long-term monitoring of transplanted cells and information on cell migration, distribution, viability, differentiation etc. To obtain information at the single cell level in vivo, we need to use model organisms, amenable to imaging. We will here use our well-established chick embryo model either by direct injection of cells in the vasculature or organs or by exploiting the chorioallantoic membrane (CAM) model. CAM has the ability to support the growth and maintenance of live tissue/cells placed on its surface, making it an ideal bioreactor and a core resource to evaluate biological processes in regenerative medicine and cancer research. The chick embryo is not under home office regulation until Embryonic day14, thereby contributing to Replace / Reduce animal use. The limitation to date has been down to the lack of a labelling technology capable of transitioning from in vitro to in vivo due to poor sensitivity, photobleaching and toxicity. StreamBio has developed Conjugated Polymer Nanoparticles (CPNs), which are highly stable, fluorescent labelling probes immensely brighter than conventional technologies, and have an iron oxide component for MRI contrast enhancing, making them multi-modal. They are taken up by cells through endocytosis and can therefore label any cell type. Using the advanced imaging technologies at the University of Liverpool Centre for Preclinical Imaging -CPI- and Centre for Cell Imaging -CCI- for cellular and in vivo imaging; we will determine how neural undifferentiated/dedifferentiated cells interact with immune cells, the host tissue and vasculature, and how this environment impact on their identity in terms of differentiation, survival and migratory capabilities.
Project objective:
1. Optimisation of long-term cell labelling capabilities of CPNs and their suitability for multiple cell type tracking in vitro; using neural stem cells and brain tumour cells, on their own or in co-culture with immune cells. We will be testing multiple-colour labelling with the CPNs and optimise their use for cell migration tracking. Upon initial characterisation in 2D cell culture, 3D spheroid models with multiple cell types will be used to test probe multiplexing and mimic tissue organisation.
2. Short/medium-term tracking of neural stem cells and neuronal cancer cells labelled with the CPNs and injected in the chick embryo. Initially, tracking in vivo will be performed in real time upon injection. Labelled cells will be injected in the chick extra-embryonic blood vessels and directly in the brain, and followed live in the chick circulation and upon settling in the chick organs.
3. Long-term cell tracking using the CAM assay and multimodal imaging; by implanting neuronal cell types (stem cells / brain tumour cells), with immune cells, each labelled with specific probes, onto the CAM. Cell proliferation, survival/death and differentiation using fluorescence detection of the probes and immunolabelling will be monitored. Importantly, we will also be able to track cell dissemination into the chick organs by using both the fluorescence and magnetic properties of the nanoprobes. The stable fluorescence multiplexing will enable individual yet simultaneously tracking of cell types. Exploiting further the multiplexing we will label both control cells and cells treated pharmacologically / knocked-down for specific signalling pathways to obtain a mechanistic unde