AUTOMATIC CELL FATE ENGINEERING USING MICROFLUIDICS DEVICES
Lead Research Organisation:
University of Bristol
Department Name: Engineering Mathematics and Technology
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.
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.
- 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.
People |
ORCID iD |
Lucia Marucci (Principal Investigator) |
Publications
Aulicino F
(2020)
Canonical Wnt Pathway Controls mESC Self-Renewal Through Inhibition of Spontaneous Differentiation via ß-Catenin/TCF/LEF Functions
in Stem Cell Reports
Blyth M
(2023)
Numerical methods for control-based continuation of relaxation oscillations
in Nonlinear Dynamics
De Cesare I
(2021)
ChipSeg: An Automatic Tool to Segment Bacterial and Mammalian Cells Cultured in Microfluidic Devices.
in ACS omega
De Cesare I
(2022)
Control-Based Continuation: A New Approach to Prototype Synthetic Gene Networks.
in ACS synthetic biology
Gorochowski T
(2020)
Towards Engineering Biosystems with Emergent Collective Functions
Gorochowski TE
(2020)
Toward Engineering Biosystems With Emergent Collective Functions.
in Frontiers in bioengineering and biotechnology
Khazim M
(2019)
Towards automated control of embryonic stem cell pluripotency
in IFAC-PapersOnLine
Khazim M
(2021)
A Microfluidic/Microscopy-Based Platform for on-Chip Controlled Gene Expression in Mammalian Cells.
in Methods in molecular biology (Clifton, N.J.)
Landon S
(2021)
Understanding Metabolic Flux Behaviour in Whole-Cell Model Output.
in Frontiers in molecular biosciences
Landon S
(2019)
Genome-driven cell engineering review: in vivo and in silico metabolic and genome engineering.
in Essays in biochemistry
Marucci L
(2020)
Computer-Aided Whole-Cell Design: Taking a Holistic Approach by Integrating Synthetic With Systems Biology.
in Frontiers in bioengineering and biotechnology
Montes-Olivas S
(2019)
Mathematical Models of Organoid Cultures.
in Frontiers in genetics
Pedone E
(2022)
ß-catenin perturbations control differentiation programs in mouse embryonic stem cells.
in iScience
Pedone E
(2019)
Role of ß-Catenin Activation Levels and Fluctuations in Controlling Cell Fate.
in Genes
Pedone E
(2021)
Cheetah: A Computational Toolkit for Cybergenetic Control.
in ACS synthetic biology
Pedone E
(2019)
A tunable dual-input system for on-demand dynamic gene expression regulation
in Nature Communications
Postiglione L
(2018)
Regulation of Gene Expression and Signaling Pathway Activity in Mammalian Cells by Automated Microfluidics Feedback Control.
in ACS synthetic biology
Postiglione L
(2019)
A strategy for multicellular feedback control in mammalian cells
Rees J
(2018)
Designing Minimal Genomes Using Whole-Cell Models
Rees-Garbutt J
(2020)
Furthering genome design using models and algorithms
in Current Opinion in Systems Biology
Rees-Garbutt J
(2020)
Designing minimal genomes using whole-cell models.
in Nature communications
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. |
Sectors | Pharmaceuticals and Medical Biotechnology |
URL | https://www.sciencedirect.com/science/article/pii/S2405896319321238 |
Description | We have been discussing applications of cybergenetics in public engagement activities (e.g. talks at local schools). |
First Year Of Impact | 2021 |
Sector | Digital/Communication/Information Technologies (including Software),Education |
Impact Types | Cultural |
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 | 09/2017 |
End | 09/2020 |
Title | Algorithms for cell segmentation |
Description | We developed multiple algorithms for microscopy image segmentation (De Cesare et al, ACS Omega 2021; Pedone et al. ACS Synthetic Biology 2021). All code is open source |
Type Of Material | Technology assay or reagent |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | Automatic control of gene expression in mammalian cells, using online segmentation algorithms. |
URL | https://github.com/BiocomputeLab/cheetah |
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 | Inducible system to perturb gene expression |
Description | The proposed inducible system allows modulating independently gene expression and protein stability by the addition of two orthogonal drugs. |
Type Of Material | Model of mechanisms or symptoms - in vitro |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | We used the tool to demonstrate the role of signalling pathway does in embryonic stem cell fate (DOI 10.1016/j.isci.2022.103756) |
Title | New microfluidic device |
Description | We developed a new microfluid device, that enables to continuously separates viable and non-viable T-cells according to their dielectric properties |
Type Of Material | Improvements to research infrastructure |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | Not know yet (recent publication) |
URL | https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/full/10.1002/elps.202100031 |
Title | Turbidostat |
Description | We designed and engineered a turbidostat for the continuous culture of bacteria. |
Type Of Material | Improvements to research infrastructure |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | Our design is open-source and the device is low-cost, making it a technology easy to adapt and use for the community. |
Title | Algorithms for cell segmentation |
Description | We developed new algorithms for cell segmentation, based on the Otsu methods (De Cesare et al. ACS Omega 2021) and deep-learning (Pedone et al. ACS Synthetic Biology 2021). |
Type Of Material | Computer model/algorithm |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | These algorithms enabled robust automatic feedback control of living cells. |
URL | https://www.github.com/BiocomputeLab/cheetah |
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 |
Title | Full pipeline for mammalian cell control |
Description | We reported in a book chapter (Khazim et al Synthetic Gene Circuits 2021) a full protocol to perform cybergenetics experiments in mammalian cells. |
Type Of Material | Data analysis technique |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | Still to be assessed (recent publication) |
Title | Minimal Genome Design Algorithms using whole-cell models Rees-Garbutt and Chalkley et al 2019 |
Description | In the future, entire genomes tailored to specific functions and environments could be designed using computational tools. However, computational tools for genome design are currently scarce. Here we present algorithms that enable the use of design-simulate-test cycles for genome design, using genome minimisation as a proof-of-concept. Minimal genomes are ideal for this purpose as they have a simple functional assay whether the cell replicates or not. We used the first (and currently only published) whole-cell model for the bacterium Mycoplasma genitalium. Our computational design-simulate-test cycles discovered novel in-silico minimal genomes which, if biologically correct, predict in-vivo genomes smaller than JCVI-Syn3.0; a bacterium with, currently, the smallest genome that can be grown in pure culture. In the process, we identified 10 low essential genes and produced evidence for at least two Mycoplasma genitalium in-silico minimal genomes. This work brings combined computational and laboratory genome engineering a step closer. |
Type Of Material | Database/Collection of data |
Year Produced | 2019 |
Provided To Others? | Yes |
URL | https://data.bris.ac.uk/data/dataset/1jj0fszzrx9qf2ldcz654qp454/ |
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 |
Title | Computational toolkit for cybergenetic control |
Description | The computational platform allows integrated online cell segmentation and control. |
Type Of Technology | Software |
Year Produced | 2021 |
Open Source License? | Yes |
Impact | The code is open-source and easy to modify/adapt for other cell lines and microscopy settings. |
URL | https://pubs.acs.org/doi/abs/10.1021/acssynbio.0c00463 |
Description | Invited Seminar, Sheffield University, January 2023 (in person) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Postgraduate students |
Results and Impact | Invited talk to the Department of Chemical and Biological Engineering; attended by PhD students, postdocs and academics. |
Year(s) Of Engagement Activity | 2023 |
Description | Invited talk, EMBO workshop "Network inference in biology and disease" |
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 | Workshop bringing together interdisciplinary researchers in biotechnology, bioinformatics, systems biology and synthetic biology. |
Year(s) Of Engagement Activity | 2019 |
URL | https://meetings.embo.org/event/19-network-inference |
Description | Mammalian Synthetic Biology Workshop 2022, Edinburgh (UK) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Major international conference in mammalian cell synthetic biology. Antonella La Regina (research associate in my group) gave a talk about cybergenetics applications in stem cells. |
Year(s) Of Engagement Activity | 2022 |
URL | http://mammalian-synbio.org/2022 |
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, 2021 Virtual International Mammalian Synthetic Biology Workshop |
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 |
Year(s) Of Engagement Activity | 2021 |
URL | http://mammalian-synbio.org/2021 |
Description | Oral Presentation, Dynamics Days Digital 2020 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Conference attended mainly by academics |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.danieleavitabile.com/ddd2020/ |
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 |
Description | Synthetic Biology UK 2022, keynote talk, Newcastle (UK) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Invited talk to the major synthetic biology conference in the UK. |
Year(s) Of Engagement Activity | 2022 |
URL | https://www.eventsforce.net/biochemsoc/frontend/reg/thome.csp?pageID=65741&eventID=132&traceRedir=2 |
Description | Talk at "Multi-agent modeling meets synthetic biology" workshop |
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 | Workshop held at the University of Bristol, UK and funded by BrisSynBio, a BBSRC/EPSRC Synthetic Biology Research Centre (Grant No. BB/L01386X/1). It was attended by key UK experts in agent-based modelling of biological systems; it sparked discussions about the use of multiscale frameworks in synthetic biology. |
Year(s) Of Engagement Activity | 2019 |
URL | https://doi.org/10.3389/fbioe.2020.00705 |