Controlling muscle function of stem-cell derived myofibres with optogenetic probes

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

Abstract

Muscles execute all movements and are the main drivers of animal behaviour. While muscle development, physiology and regeneration are well understood, the recreation of muscle from its constituent cellular and extracellular components is still in its infancy. We propose a project which will explore new ways to model muscle function with 3D-printed constructs assembled from pluripotent stem cell-derived muscle cells and artificial scaffolds. To control contractions, we will emulate synaptic input with optogenetic. The aim of the project is to develop muscle constructs and apply the technology to study muscle degeneration and explore bioengineered muscle as bio-actuators in soft robots.

We offer a multidisciplinary Ph.D. project which will encompass stem cell biology, optogenetics, biomaterial science and engineering, with the aim of creating artificial skeletal muscle which can be controlled by light pulses. To this end, the student will genetically modify stem cells with transposition and gene targeting, develop methods to derive myoblasts from mouse and human pluripotent stem cells and magnetically purify the cells. We will induce cell fusion to assemble aligned myofibers in 3D-scaffolds composed of synthetic and/or biological polymers by 3D printing and electrospinning, and then control myofibre contraction with light through genetically encoded photo-sensors such as channelrhodopsin-2. This external control system removes the neural component from the experimental model and replaces synapse with optical input. Thus, neuromuscular connectivity is not necessary for the artificial muscle to function, and this allows direct control of muscle function and greatly simplifies the cellular architecture of the in vitro model.
Once the optogenetic muscle constructs have been developed, the student will then explore two different applications of the technology:
1) The student will investigate the consequences of genetic mutations which affect the structural integrity of muscle, such as loss-of-function and gain-of-function defects in the dystrophin gene. Normal and mutant muscle will be entrained with long-term rhythmic light stimulation, and the effects on muscle integrity and homeostasis will be assessed. This system will serve as a test case for studying muscle function and dysfunction in vitro.
2) The student will assemble a (reductionist) musculoskeletal system, for example one composed of two antagonistic muscle constructs pulling a rod attached to an artificial hinge joint into opposite directions. Movement in this system will then be controlled by specific optical activation of one of
the two muscle constructs. Such an arrangement would simulate motor behaviour in vitro and might serve as a precursor to more complex semi-biological robotic systems.

Publications

10 25 50

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M009513/1 01/10/2015 30/09/2023
1902542 Studentship BB/M009513/1 01/10/2017 30/09/2021 Aimee Cheesbrough
 
Description Skeletal muscle is a highly organised hierarchical tissue, responsible for driving animal movement and behaviour. Seemingly simple skeletal movements are governed by an underlying network of complex neuromuscular interactions. Healthy skeletal muscle has an inherent ability to strengthen in response to mechanical load, and regenerate in response to damage; however, normal muscle function and homeostasis can be compromised as a result of genetic, metabolic and age-related disease. This can lead to more rapid degeneration-regeneration cycles, exhausting the satellite cell pool, and causing subsequent muscle wasting and fatigue. In order to fully understand the mechanisms that regulate muscle homeostasis, physiologically and functionally relevant models are required. Commonly used animal models are both cost- and time-intensive, whilst 2D cell culture assays fail to capture the 3D arrangement of cells and their surrounding extra-cellular matrix, limiting functional-assay studies. Through the development of a functional in vitro model of the muscular system, this project aims to bridge the gap between these common approaches, and enable the study of muscle homeostasis in healthy and compromised phenotypes. This project combines muscle biology, stem cell technology and biomaterial science in the development of a bio-hybrid tool comprising embryonic stem cell-derived muscle fibres and electrospun nanonfibre sheets anchored with PDMS micro-pillars. Whilst also supporting the contractile nature of the muscle fibres, the synthetic components provide a tendon-like structure which will be used to convert the function from the biological tissue, into a controlled and measurable output. Targeted activation of specific cell populations will be made possible using two-colour optogenetics, providing the tools to build more complex antagonistic systems that could emulate an in vivo hinge joint.

The final model brings together many different components, each of which have been investigated and tested in the initial stages of the project. Firstly, I have successfully developed a method to derive myoblast cells from mouse embryonic stem cells. Using inducible cassette exchange (ICE), we have inserted a doxycycline-inducible transgene (MyoD) into mouse ESCs. A tetracycline-response element (TRE), located upstream of the transgene, is activated by exposure to doxycycline, which enables efficient expression of MyoD, and generates myoblasts after 7 days in culture. Genetic integration of a protease-resistant surface marker, CD14, under the muscle-specific transcription factor Myogenin, enables myoblasts enrichment of up to 90% in both ICC stains and flow cytometry analyses. In traditional 2D culture, these sorted myoblasts further differentiate and fuse to become myotubes after 9 days, and mature myofibres after 12-13 days, at which point, their inherent contractile nature makes further culture challenging. As such, alternative culture substrates are necessary which support muscle fibres in their contractile physiological environment. Using photolithography techniques at London Centre for Nanotechnology (LCN), I have created a mould which enables me to systematically produce arrays of micro-culture wells with flexible PDMS micropillars. Using AutoCAD, I designed photomasks with the described array etched onto them, and used these masks to expose regions of photo-curable resin (SU8) onto a silicon wafer. The wafer was then silanized, coated with PDMS and cured. The individual microdevices were then cut and glued to tissue culture plates. The PDMS pillars within these micro-devices provide a tendon-like structure for supporting the contractile muscle in culture. One of the key challenges in generating mature myofibres, is insuring that myoblasts align with one another, as they do in the body. I have therefore developed novel polyurethane based aligned electrospun nanofibres (POSS-EDS-PU), that provide an adhesive, elastic culture substrate, and guide the alignment of the myofibres. This experiment is in its early stages, but initial data looks promising and will be incorporated into the micro-wells. Finally, we aim to use this technology to create a more complex reductionist system that emulates an in vivo hinge joint. This requires independent excitation of cell populations (muscle constructs) within the same culture dish. As such I have stably integrated both blue and red light Channelrhodopsins (ChR2 and Chrimson) into the mESC, for controlling contractile behaviour by optogenetic stimulation.
Exploitation Route The award is still open and I am roughly half way through the PhD project. I will now begin to bring all these individual components together into one system, which could then be used to investigate muscle fibre homeostasis in healthy and diseased tissue. I have begun a collaboration with Dr Yung-Yao Lin at QMUL, who specialises in modelling muscular dystrophies (MD). He had provided iPSCs with mutations in the FKRP gene, which is thought to disrupt functional glycosylation of alpha-dystroglycan, a process which is critical for maintaining muscle integrity. We will collaborate on incorporating these cells with these novel biomaterial methods, as a potential tool for identifying MD drug targets in the future.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
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.