AUTOMATIC CELL FATE ENGINEERING USING MICROFLUIDICS DEVICES

Stem cells are pluripotent cells that can both proliferate indefinitely producing cells identical to them, and specialise into more mature cells types. In adults, stem cells have a repair function in case of damage; adult stem cells are currently used in medical therapy. The major limitation of adult stem cells' medical applications is their low availability, and the difficulty to expand them in culture.

Such issues were thought to be overcome thanks to the astonishing discovery of reprogramming by the Nobel Prize-winning Shinya Yamanaka: differentiated (i.e. somatic) cells can be programmed back to a stem-like state, obtaining the so-called induced Pluripotent Stem Cells (iPSCs). iPSCs can be subsequently converted into any cell type, to be used for regenerative and personalised medicine purposes. In Japan, the first clinical trial using iPSC-derived cells in humans is on going to cure age-related macular degeneration.

iPSC therapy still faces, however, major challenges: it is difficult to reprogram somatic cells and maintain iPSCs in the pluripotent state; also, iPSC differentiation is often inefficient.

In this research, we aim at applying state-of-the-art Synthetic Biology and Control Engineering tools to automatize and optimise the manufacturing of iPSC-derived cells. We will prove, using mouse cell lines, that each of the 3 mentioned challenges can be addressed if, while providing inputs that trigger pluripotency or differentiation, cells are continuously observed and inputs are consequently "adjusted" to obtain the target phenotype. This closed-loop strategy will be implemented by means of microfluidics and microscopy, that allow monitoring in real-time living cells, comparing relevant cellular outputs to the target one and applying control algorithms that allow acting on the cells to minimise the error.

While proving that, by "closing the loop", it is possible to automatically control stem cell fate, we will provide a platform that allows, at the end of the experiment, to retrieve from the microfluidics device the desired cell type with high efficiency and reproducibility.

The research in this proposal takes a multidisciplinary approach to develop robust methods for steering cellular fate in the manufacturing of induced Pluripotent Stem Cell- (iPSC-) derived cells, and will have an impact on:
- Academic community
- Biomedical industry
- General public
- Research personnel involved in this project

Academic community
Academics benefiting directly from the success of this proposal include scientists in the Synthetic Biology, Systems Biology, Control Engineering and Biomedical Science communities.
Achieving targeted gene expression regulation in mammalian cells is a great challenge in Synthetic Biology. While newly developed technologies (i.e. CRISPR-Cas9 systems) enable efficient genome editing, fine-tune regulation of complex endogenous regulations with open-loop strategies lacks precision and robustness to noise. We will provide a complete computational/experimental framework based on closed-loop and microfluidics, which could be used for the characterisation of synthetic components and their interactions with endogenous ones.
Researchers in Systems Biology will be provided with mathematical models of cellular response to drugs in highly relevant biological processes (e.g. reprogramming and differentiation), and with segmentation and deep learning algorithms for microscopy images quantification and classification.
We will compare the performance of different algorithms for the implementation of complex control tasks in living cells; these results will be beneficial for the Control Engineering community, and will fuel novel research for the development of stochastic and single-cell control strategies for living mammalian cells.
The proposed research has the potential to have implications in regenerative medicine: although our research is focused on the reprogramming, pluripotency maintenance and differentiation of mouse cells, our pipeline could be extended to the engineering of human stem cell fate, which would be highly relevant for Biomedical scientists.

Biomedical industry
The demand for stem cells-related products is rapidly growing. The work proposed here, exploiting closed-loop strategies and microfluidics technologies, has the potential to provide basis for refined reprogramming, pluripotency culture and differentiation protocols , which would be highly appealing for companies who provide stem cell related products and, more generally, for the the biomedical industry. Possible applications of our computational/experimental platform include the automatic screening of synthetic compounds targeting endogenous components of pathways involved in pluripotency and differentiation.

General public
Regenerative medicine and Synthetic Biology are topics of great interest and debate for general public, given potential applications in gene therapy and ethical concerns. We believe that sharing research ideas and results with general public is an honest and clear way is a fundamental part of the scientist work. Therefore, we will disseminate findings and, more generally, research ideas behind the field of Synthetic Biology by taking part to public engagement activities organised by the University of Bristol and the Institute of the project partner.

Research personnel involved in this project
The two appointed PDRAs will gain a number of cutting edge skills that will help them in their future academic or industrial career. A benefit of this proposal is that the PI is experienced in working in both the experimental and the computational communities, and currently mentors interdisciplinary researchers.
If funded, this application will provide the PI with a framework for developing new capabilities for future research funding opportunities from UK and EU Research Councils, to inform her undergraduate and postgraduate teaching and to develop new links with leading research institutes and the biomedical industry.