Periodic Reporting for period 3 - HISTOID (A Human iPS Cell-Derived Artificial Skeletal Muscle for Regenerative Medicine, Disease Modelling and Drug Screening) Reporting period: 2021-09-01 to 2023-02-28 Summary of the context and overall objectives of the project Skeletal muscle is the most abundant human tissue and its regenerative capacity is compromised in several neuromuscular and musculoskeletal diseases, such as muscular dystrophies. Muscle diseases are amongst the most frequent causes of disability in Europe, affecting a large socioeconomic spectrum. The most severe muscular dystrophies (often paediatric and unfortunately still incurable) pose also serious economic burdens on health services and families, due repeated hospital admissions, surgery, home care, physiotherapy and devices required to counteract muscle wasting. Therefore, developing reliable in vitro models of human skeletal muscle would be instrumental for studying disease mechanisms and developing novel therapies. However, current models are challenged by the limited lifespan and differentiation ability of biopsy-derived muscle-making cells (known as myoblasts) and by the low-fidelity of standard cell culture techniques.My work has pioneered the use of human stem cells known as induced pluripotent stem cells (iPSCs) to generate unlimited genetically-corrected transplantable muscle cells. In the HISTOID project we are exploiting this technology together with biocompatible materials to develop three dimensional, iPSC-derived, patient-specific artificial muscles to study human muscle disease and regeneration, as well as to develop new therapies for severe muscle diseases.The project is currently being developed in two phases. First, we developed the iPSC-derived muscle in vitro, introducing cell types and stimuli relevant for skeletal muscle function. In the second phase we will exploit the artificial muscles for regenerative medicine and drug development. Specifically, we will investigate the artificial muscle potential for tissue replacement and then model different muscular dystrophies in vitro to screen drugs with therapeutic relevance. Finally, we will combine the tools and knowledge developed in the two aforementioned areas into a novel platform to optimise skeletal muscle gene and cell therapies. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far From the start of the project we have made significant progress in all Aims planned for Work Package 1 (i.e. Aims 1,2,3), including also progress in WP2 on using the iPSC-derived artificial muscle to study and repair skeletal muscle. Specifically: Aim 1 - iPS Cell Differentiation into Lineages Present in Human Skeletal Muscle (year 1).We have successfully generated several iPSC-derived cell lines relevant for muscle physiology using our and other established methods. With this approach we generated iPSC-derived muscle fibres, muscle progenitor cells, blood vessel cells (i.e. endothelial cells and pericytes) and motoneurons from the very same patients. We are also optimising methods to generate fibro-adipogenic cells from iPSCs, as they are relevant to support muscle regeneration.Aim 2 – Three Dimensional Assembly of Differentiated Human iPS Cells into Biomaterials (year 1-2).We have observed consistent formation of muscle fibres upon differentiation of iPSCs in human biocompatible material (i.e. fibrin) in 3D. Notably, we successfully generated artificial muscles containing up to four cell types from the very same patient, all iPSC-derived, i.e.: muscle fibres, endothelial cells, pericytes and motoneurons.Aim 3 – Morphological and Functional Characterisation of the Human Artificial Muscle Tissue (year 2).Histology, immunostaining, gene expression and western blot analyses have been performed at different stages of maturation of the artificial muscles and showed presence of hallmarks of mature muscle such as sarcomeric and dystrophin-associated proteins. Formation of neuromuscular junctions has been demonstrated by labelling postsynaptic acetylcholine receptors with fluorochrome-conjugated alpha-bungarotoxin. Vascular structures have bees shown by CD31 immunostaining for endothelium and SM22-positive, GFP expressing pericytes were also identified in the 3D muscles. Muscle satellite-like stem cells were also identified by Pax7 staining in the 3D muscle generated using a transgene-free iPSC differentiation method. Sarcomeres were confirmed by electron microscopy and their function was assessed by detection of calcium transients following caffeine administration.On top of the 3 aforementioned Aims constituting the core of this first reporting period, we have also made significant progress in aims 4 and 5. Specifically, we have completed important experiments assessing muscle replacement in volumetric tissue loss in vivo in mice, as well as reported the very first disease modelling platform for muscular dystrophy based upon an iPSC-derived 3D artificial muscle (Maffioletti SM et al., Cell Reports 2018; Steele-Stallard HB et al., Front Physiol 2018). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We have made significant progress beyond the state of the art in modelling skeletal muscle laminopathies using the HISTOID 3D muscle platform, identifying reproducible mutation-specific morphological phenotypic readouts (i.e. nuclear shape abnormalities). These readouts have been validated across multiple cell lines and 3D muscles, including a cell line which is targetable with therapeutic antisense oligonucleotides (Steele-Stallard HB et al., Front Physiol 2018). This part of the HISTOID is progressing remarkably well, with expansion into gene therapy-/editing-based strategies to target also other mutations. We have also generated new iPSC line from patients with Duchenne muscular dystrophy which can be used to screen new therapeutics in 3D muscles (Ferrari G et al., Stem Cell Res 2020). Finally, we have started to generate preliminary data for the application of the 3D muscle platform to screen gene therapy vectors (an area to be further expanded in WP2, Aim 6). Overall, despite the challenges posed by the Covid-19 pandemic, we have made significant progress in the development of the HISTOID project.