Periodic Reporting for period 2 - HISTOID (A Human iPS Cell-Derived Artificial Skeletal Muscle for Regenerative Medicine, Disease Modelling and Drug Screening)
Reporting period: 2020-03-01 to 2021-08-31
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.
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).