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).
