AUTOMATIC CELL FATE ENGINEERING USING MICROFLUIDICS DEVICES

Lead Research Organisation: University of Bristol
Department Name: Engineering Mathematics

Abstract

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.

Planned Impact

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.
 
Description Thanks to this grant we are performing experiments to directly control stem cell identity, for the development of improved pluripotency and differentiation protocols.
Exploitation Route We were able to control the expression of pluripotency genes in mouse embryonic stem cells by means of a microfluidic platform, segmentation and control algorithms. and motor-controlled syringes.

We now need to extend the capability of the current microscopy/microfluidics platform to enable longer experiments and perturbations with a higher number of drugs. Recent additional funding (EPSRC Fellowship to LM, EP/S01876X/1) will support this research.

If we succeed in developing superior protocols for cell differentiation, the new protocols could be used by other groups. Also, upon publication, we will make the code for mammalian cell segmentation and control available.
Sectors Pharmaceuticals and Medical Biotechnology

URL https://www.sciencedirect.com/science/article/pii/S2405896319321238
 
Description Horizon 2020 FET-Open action "Control Engineering of Biological Systems for Reliable Synthetic Biology Applications"
Amount € 3,000,000 (EUR)
Funding ID Grant agreement ID: 766840 
Organisation European Commission 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 10/2017 
End 09/2020
 
Title Automatic feedback control of gene expression in mammalian cells 
Description We developed a microfluidics/microscopy platform to measure and control gene expression in mammalian cells. This, in turns, required specialised software for online cell segmentation, control and actuation. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2018 
Provided To Others? Yes  
Impact The methodology can be used to track dynamics in living mammalian cells, and to automatically infer improved protocols. Results published in Postiglione et al, ACS Synth. Biol. 2018, 7, 11, 2558-2565. 
URL https://pubs.acs.org/doi/abs/10.1021/acssynbio.8b00235
 
Title Data associated to publication (Pedone et al, Nature Communications 10, 4481 (2019)) 
Description All data generated in the publication are included. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact The tools developed might support generating more precise and complex tools to manipulate gene expression in living cells. 
URL https://www.nature.com/articles/s41467-019-12329-9#data-availability
 
Description Segmentation and control of mammalian stem cells 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Country Germany 
Sector Academic/University 
PI Contribution My team is generating data of mammalian stem cell differentiation, using a microfluidics/microscopy platform for cell imaging and control.
Collaborator Contribution The partner is currently supporting online cell segmentation.
Impact The collaboration is multidisciplinary and still active, so there are no outputs yet.
Start Year 2019
 
Description Open Days Engineering, Bristol University (UK) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Open Days for A level students, which involved presentations by PhD students and PDRAs, and demos.
Year(s) Of Engagement Activity 2017,2018,2019,2020
 
Description Oral Presentation, London Stem Cells Network symposium, Francis Crick Institute, 2018, London (UK) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Specialised Stem Cell Biology conference.
Year(s) Of Engagement Activity 2018
URL https://lscn.crick.ac.uk/?page_id=2
 
Description Oral Presentation, Research Conference, 18th European Control Conference (ECC) 2019, Naples (Italy). 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Interdisciplinary conference about principles and applications of control engineering.
Year(s) Of Engagement Activity 2019
URL https://ecc19.eu/
 
Description Oral Presentation, Research Conference, Foundations of Systems Biology in Engineering (FOSBE) 2019 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Research conference on applications of control engineering in Systems and Synthetic Biology; interdisciplinary audience.
Year(s) Of Engagement Activity 2019
URL http://fosbe2019.ai2.upv.es/
 
Description Oral Presentation, Synthetic Biology UK (SBUK), Bristol 2018 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Most relevant Synthetic Biology conference in UK; interdisciplinary audience.
Year(s) Of Engagement Activity 2018
URL https://www.eventsforce.net/biochemsoc/frontend/reg/thome.csp?pageID=18045&eventID=43&traceRedir=2
 
Description Oral Presentation, Synthetic Biology: Engineering, Evolution & Design (SEED), 2019, New York (US) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Interdisciplinary conference on Synthetic Biology.
Year(s) Of Engagement Activity 2019
URL http://synbioconference.org/2019