Modelling peripheral axonal pathology of ALS in vitro - Assembly of optogenetic neuromuscular circuits from pluripotent stem cells

Lead Research Organisation: King's College London
Department Name: Genetics and Molecular Medicine


Amyotrophic Lateral Sclerosis (ALS) is a medical condition which affects motor neurons, the type of nerve cell which connects the brain to muscle. ALS leads to paralysis, so that affected individuals can no longer walk, speak or breathe. The disease is both quite common and currently incurable, and therefore many scientists study its causes and possible therapies. As many manipulations required to investigate ALS cannot be carried out in patients, experimental animals, mostly mice and rats, are used in most studies. These animals are often genetically modified such that they develop a disease which is similar to the one seen in humans. This approach uses a large number of animals and is expensive and time-consuming, and therefore not suitable to systematically study hereditary traits which may cause ALS.
We propose to develop an alternative way of investigating ALS, which will be based on complex, life-like artificial tissues grown directly from cultured human stem cells. The artificial tissues will resemble nerve tissue (motor neurons and accessory cells called 'glia') and muscle. They will be cultured in a compartmentalized biochip designed and manufactured for this project. In this device, the two tissues will be segregated - as they are in humans and animals-, and the motor neurons will connect to muscle through nerve fibres and control muscle contractions. The cells in the device will contain biological optical probes that allow us to feed instructions into the cells on the biochip and read information out though fluorescent light signals. We will test the validity of the new experimental system by first modelling the degeneration of nerves and the loss of communication between nerves and muscle. Then, we will use this 'ALS in a dish' model to investigate why different forms of ALS result in different patterns of degeneration, how genetic traits protect some people from developing ALS, and how such protection may be mimicked with chemical compounds.
We think that this tissue culture model could allow scientist to discover ALS drugs faster, cheaper and without the use of animal experimentation.

Technical Summary

We propose to develop an in vitro model of Amyotrophic Lateral Sclerosis (ALS) and neuromuscular circuitry, based on sorted pluripotent stem cell-derived motor neurons, astrocytes and myofibres, which are co-cultured in a compartmentalized tissue culture device. We plan to investigate the disease mechanism of ALS, with a focus on peripheral synaptic and axonal cellular pathology. To this end, we will equip human induced pluripotent stem cells carrying disease-associated mutations in the genes SOD1, TARDBP or C9ORF72 with genetically encoded optogenetic actuators and optical probes which will allow us to stimulate the cells, read their electrical activity and trace cellular features with live imaging. The optogenetic stem cell-motor neurons will be activated with light and used to control the myofibres connected to them through neuromuscular junctions (NMJs). We will map different ALS-associated mutations to specific patterns of early cellular degeneration, which may be initiated either centrally or peripherally. We will use this in vitro model to study the impact of different neural activity patterns, modifier genes, and small molecule compounds on early pathological features, in particular abnormal NMJs and defective axonal transport. This may enable us to pinpoint potential therapeutic pathways, and, in the long-term, develop ALS drugs which are specifically customized to the individual genotypes and phenotypes of ALS patients.

Planned Impact

Exploitation and Application (year 3-4):
We intend to develop a new type of stem cell-based in vitro model of neuromuscular circuits and neuromuscular disease, which, long-term, may be used for drug discovery. This research promises to create valuable intellectual property (IP) as such a tissue culture system that would be highly desirable for pharmaceutical companies interested in developing treatments for neuromuscular disease, such as ALS. The technology transfer teams of KCL and National University of Singapore (NUS) will engage and liaise with the PIs to identify new IP should it arise. King's Commercialisation Institute has extensive experience in IP management and exploitation and has established working relationships with reputable IP law firms. IP arising shall be owned by the host institutions of the applicants and project partners. Commercial partners will be sought at an early stage following the filing of any patent applications.

Advancing Training (year 1-4):
The proposed grouping (PIs and project partners) have considerable expertise in the molecular, cellular and morphological analyses of neural and muscle tissue, which will provide excellent training opportunities for the named postdoctoral researcher, Dr Machado, in a variety of skill sets. Dr Machado will be involved in the supervision of lab projects that are part of the Ph.D. programs at KCL. In this way, we will provide an environment that will contribute to the training of scientists, and which will facilitate the development of skills to acquire her ability to manage and supervise lab projects. Furthermore, Dr Lieberam is co-supervising a graduate student, Miss Perrine Pluchon, for a joined (separately funded) Ph.D. thesis in his group and the group of Dr Virgile Viasnoff, NUS. Miss Pluchon is working with Dr Viasnoff on optimizing the material science aspects of the compartmentalized tissue culture device, and will move to Dr Lieberam's group in London in November 2015 to help adapt the system to cultures of sorted ESC-derived neural cells and myofibres. Participating in this project provides a unique training opportunity from her, and might allow her to learn a wide variety of scientific concepts, experimental methods and manufacturing techniques in stem cell biology and engineering.

Communication and Dissemination of Data (year 3-4):
We support the RCUK's policy to make the research data generated via their funding to be available to the community in as timely a manner as possible. The proposed project will generate a number of different data types, which will all stored in industry standard formats to maximise data portability. Data will be automatically archived and centrally collected in Dr Lieberam's group (see Data Management Plan). Our intention is to make our data available primarily through peer-reviewed scientific journals that can be accessed through PubMed, allowing our main findings to be monitored for accuracy and content. In line with current policy we will also ensure that data are published in journals with an Open-Access policy of not more than 12 months ('Green' option). Whenever possible, we will choose immediate unrestricted access ('Gold' option). Any reagents produced will be made freely available to the scientific community immediately on publication through repositories such as Addgene. We plan to publish and/or present our data at meetings.

Public Understanding of Science (year 1-4):
Throughout the project we will engage with school students and support teachers. Dr. Lieberam supports the educational charity In2ScienceUK and is accepting school students from low-income backgrounds for a week of work experience in his laboratory to encourage them pursue a career in science. We are committed to the idea that an engagement in teaching and education at different levels is an important aspect of scientific practice.


10 25 50
Description Proximity to Discovery Industry Engagement Fund
Amount £29,960 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2018 
End 06/2018
Title GFAP::CD14 ES cells 
Description The GFAP::CD14 ES cells (clone H6) carry a stable transgene that allows the isolation of astrocytes directly differentiated from mouse ES cells by magnetic cell sorting 
Type Of Material Cell line 
Year Produced 2014 
Provided To Others? Yes  
Impact Together with the Mnx1::CD14-IRES-GFP ES cell lines, this line allows the generation of CNS-like neuron/glia cocultures to simulate neural patterning/neural circuitry in vitro. The system does not require the use of experimental animals, but closely resembles primary neural cultures. 
Title Hb9::CD14-IRES-GFP mouse ES cells 
Description This mouse ES cell line allows the magnetic isolation of motor neurons directly differentiated in vitro. The surface tag used, CD14, is superior to those previously available. We will make the reagent available (through UK Stem Cell Bank or other distributors) once it is published. 
Type Of Material Cell line 
Year Produced 2014 
Provided To Others? Yes  
Impact Most existing ES cell lines with motor neurons-specific promotors use fluorescent markers, which require flow cytometers for cell sorting (costs: GBP 100K - 300K). Magnetic cells sorting using surface tags works almost as efficiently and allows the purification of much larger numbers of cells (>10^8) at a fraction of the costs (ca. GBP 300 for the magnet + stand). Once this ES cell line is available, many more research groups will be able to afford working with ES cell derived neurons to explore neural development and neurological diseases. 
Title In vitro model of neuromuscular circuit 
Description In collaboration with Virgile Viasnoff (NUS, Singapore) and Juan Burrone (KCL), we have developed an open tissue culture device which allows the co-culture of PSC-derived motor neurons and astrocytes with myofibres in a connected compartments. We are currently preparing the re-submission of a revised manuscript to the journal Advanced Biosystems. It will be available to the public soon, probably May/June 2019. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2018 
Provided To Others? No  
Impact This device can be adapted to human iPSC and will be useful for screening drug for neuromuscular diseases, such as ALS, on an intact (reductionist) neuromuscular circuits. 
Title optogenetic PSC-myofibers 
Description Together with Prof. Song and me, Aimee is developing a culture system of optogenetically controlled ESC-myofibers. The long term goals is to i) model muscle diseases in vitro with a new type of myofiber-microdevice, and ii) assemble hybrid biological/synthetic soft robots that employ myofibers for locomotion and, long-term, may be used for microsurgery in vivo. To this end, she has generated mouse ESC clones that carry a MACS-sortable Myog::CD14 myoblasts reporter, a dox-inducible MyoD gene which transdifferentiates ESC-mesoderm into myoblasts, and optogenetic actuators. She chose two different channelrhodopsin, activated by blue and red light, respectively, to be able to independently control two antagonistic myofiber constructs in a hinge-joint-type musculo-skeletal model. Aimee has already tested the functionality of the optogenetic myofibers in a custom-build microdevices designed to stabilize contractile fibers, and she is now adapting the experimental system to human iPSCs with the aim of modeling muscle dystrophies. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2020 
Provided To Others? No  
Impact Once published, we plan to use this technology to develop a screening platform for compounds/genetic factors that revert muscle disease phenotypes for conditions such as Duchenne Muscle Dystrophy. Such a model system would be superior to existing cell culture methods and may be used by academic groups and pharma/biotech industry for drug development and the study of disease mechanism. 
Description Testing potential drugs for the treatment of ALS with an in vitro neuromuscular circuit model 
Organisation AstraZeneca
Department Astra Zeneca
Country United States 
Sector Private 
PI Contribution My student Perrine Pluchon worked at Astra Zeneca/Cambridge with support from the MRC Proximity to Discovery scheme from January to June 2018. The aim of her project at AZ is to transfer the neuromusuclar tissue culture technology she developed during her PhD thesis into a pharma industry setting and test if it is suitable for low-medium scale drug screens. We have since continued the collaboration with David Baker (AZ) and will adapt our neuromuscular model to high-medium throughput screening platforms.
Collaborator Contribution Astra Zeneca will provide lab space and access to proprietary chemical compounds. We also have access to additional ALS candidate drugs in our lab at KCL through an Open Innovation partnership with AZ.
Impact This collaboration started only recently.
Start Year 2017
Description Internship for GSCE-level students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact My group participated in a scheme organized by the charity In2Science UK that provides 2-week lab placements to gifted students from underprivileged backgrounds. We were impressed by the interest and enthusiasm of the student who worked with us and plan to host more students from this scheme in the future.

The student who worked with us, Adam, gave an excellent presentation to other In2Science UK students, and, hopefully, will be motivated to pursue a career in Science or Medicine in the future.
Year(s) Of Engagement Activity 2012,2013
Description Teaching material for High School Science classes 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact I have assisted Science in the Classroom (, an educational resource published by Science/AAAS, in creating an extensively annotated version of the Bryson et al. paper and a set of data analysis tasks for high school students, which will serve as an educational tool for Science teaching in classrooms in the USA and elsewhere.
Year(s) Of Engagement Activity 2014