Application of single cell metabolite profiling to optimisation of stem cell bioprocessing

The potential of stem cells as treatments for disease appears in the public arena with increasing frequency. Fundamentally, these are cells that may have the ability to be switched to take on the function of cells within critical roles in the body. Consequently, when the normal cells are damaged, as in heart attacks or paracetamol-induced liver damage, the stem cells should have the ability, as a transplant, to take up the function of the heart or liver cells. The scope exists for stem cell treatments to cure/improve the well-being of patients suffering from life-threatening/-changing conditions. Tremendous advances have been made but a number of key hurdles must be overcome before there is a more complete realisation of the undisputed potential. Chief amongst these is the fact that stem cells do have the potential to take on the functions of many different cell types and the control of this process is not fully understood. Placing this in to the context of our present proposal, the loss of insulin-production by cells of the pancreas generates diabetes. This can be treated by insulin injection, with all the consequences for monitoring and life-style, but the possibility of transplantation of insulin-producing cells to patients offers a vision of cure, rather than therapy. Hence, the key challenge is to understand the processes that control the change from stem cell to a pancreatic cell. This is against a background of stem cells undergoing changes towards many cell types - nerve, liver, muscle, kidney, heart - and generating a mixture of cell types simultaneously. Transplantation of a mixture of cells, rather than the insulin-secreting cell would not be effective. This aspect remains a major limitation for the potential of stem cell therapy to become a reality and it is the focus for this proposal. Our proposal will apply novel approaches to look at stem cells as individual cells using our pancreatic cell lines as a powerful model system. The reason this work is important is that it aims to take "fingerprints" of the metabolism of each cell as an individual entity, enabling us to relate a "fingerprint" with the controlling events that determine if a cell becomes nerve, liver, pancreas etc. The information will aid us to identify processes for maintenance of cells in whatever functional state is desired and to select for cells with a particular functional state. This type of processes can be applied to large-scale generation of cells with defined function - taking the production of cells to the commercial level that will move us towards manufacture of cells in sufficient quantity, as well as quality, to use as real-life medicines. Our approach is one that is unique due to the collaborative positioning of research groups with disruptive new technologies and model cell lines for effective development of a more general technology.

This project seeks to define the metabolic profile that is characteristic of stem cells progressing towards distinct cellular fates and profiles that are indicative of cells at different stages of differentiation. The cellular model of study - a panel of human pancreatic progenitor cell (PPC) lines - undergoes a progressive shift in differentiation status towards a glucose-sensitive insulin-secreting phenotype. Our focus is at single cell level - to remove the complexity of heterogeneity from understanding the metabolic profile of a cell with specific phenotype within a complex cellular population. We shall combine single cell imaging with fluorescent reporters of gene expression/cell surface markers of differentiation stage with application of single cell spectroscopic assessment of metabolic status (Secondary Ion MS, SIMS/matrix-assisted laser desorption ionization, MALDI/Raman) to define the metabolic status associated with the pathway of cell lineage fate. Following characterisation of phenotype-specific metabolic profiles, a major aim within the programme is to optimize the single cell non-destructive assessment (Renishaw InVia Raman microscope) to provide on-line, real-time characterisation of phenotype. Projecting our goals towards industrial bioprocessing of cellular products, we couple our prior work on development of media and feed regimes to optimize CHO cell bioprocesses to modeling the most effective media conditions to either maintain cells in an un-differentiated state (for culture expansion) or to progress towards a desired cellular fate. We address very important questions towards improvement of cellular products at commercial scale using novel approaches. The outcome of this project will have implications for, at least, diabetes therapy but will provide a model and way of thinking that has application to directed manipulation of other stem cell and regenerative medicine products.

The research seeks to describe, and understand, the heterogeneity of stem cell populations at molecular level. We have selected a stem cell model that has vast significance for the future potential cure of diabetes but which also serves as a model for the general progression of stem cells towards a specific functional fate. The promise of stem cells as "cellular medicines" that will cure otherwise intractable conditions has been widely voiced for the past decade and more. The complexity, and heterogeneity, of precursor stem cell populations and the limited (complete) differentiation towards a defined cell fate means that cells as therapeutic agents have not reached the maturity of product quality that would satisfy the regulatory authorities. As such we await breakthroughs in understanding and approach to generate therapeutically-acceptable cellular medicines at a significant level.
Our project addresses exactly this point. We are asking the fundamental question about how individual cells in a complex and heterogeneous mixture can be characterised, how they vary during differentiation and how, with that knowledge, we can use the natural processes associated with differentiation to favour the production of cells of specified phenotype. With a metabolic handle of cell phenotype and a wide ability to manipulate culture media and environment conditions, we aim to develop a platform (through single cell profiling) to relate various aspects of cellular phenotype (differentiation-specific endogenous and reporter gene expression, cellular metabolism) to generate enriched and selective cell fates. We have brought together a unique team and this is focused around novel metabolite profile at single cell level within a new model of pancreatic stem cells which we have shown to differentiation towards a beta-cell phenotype (insulin expression).
Fundamentally, the project leads to many interested parties. Diabetes and a "permanent" cure by functional cell transplantation is one direct link from our work but equally the approach can be seen to link to generation of cells with any specific fate and so is of relevance to a wide aspect of human health. Commercially, industrial concerns need to generate cell products of appropriate quantity and quality (phenotype). Understanding environmental processes of culture that enable bulking up of cells of undifferentiated phenotype and then switching when appropriate to a desired functional phenotype presents a major, and as yet unsolved, limit to wider acceptance of stem cell-derived cellular therapies. Pioneering approaches to resolve cellular phenotype as a readily-measured cellular characteristic, one that can be controlled and set by the culture environment, offer new directions for improved cellular therapeutics.
From a broader academic viewpoint, studies at single cell level present an important part of the toollbox for understanding the fundamentals of integrated cell function of significance to the control (and mis-functioning) of all facets of basic cellular functions, from transcriptional control to growth and survival. Our project is part of the development of that understanding of the cellular "system" and single cell level and can be seen as part of that thematic research area.
In summary, our project has benefits for basic scientific understanding of the mammalian cell system, health implications in relation to the desire to generate more effective cellular therapeutics and commercial relevance in aiding industrial concerns achieve the promise of cellular therapeutics as future medicines.