Human induced pluripotent stem cells for skeletal muscle diseases

Regenerative medicine along with tissue engineering represent two closely related fields leading promising advances for the treatment of numerous musculoskeletal diseases and injuries. Nevertheless, new efforts are urgently needed to design a successful therapeutic approach for muscular disorders, aiming at identifying a functional stem cell population and biomaterial scaffolds in which cells and growth factors could be embedded. In this context, recent studies have suggested that reprogramming of somatic cells by defined transcription factors into induced pluripotent stem cells (iPS), as source for generating autologous muscle progenitor cells (MPs), overcomes several limitations related to adult myoblast therapy. The prospect of an unlimited cell source combined with properties such as a more proliferative capacity in vitro, suggesting a better regenerative capacity in vivo models, indicates that iPS could be a promising candidate for stem cell therapy to regenerate skeletal muscle. iPS have been shown to retain specific features that are remnants of epigenomes and transcriptomes of the donor tissue termed ‘epigenetic memory’. Given to these findings, during the first part of the present study, we generated iPS derived from skin fibroblasts and pericytes (known to have a remarkable myogenic capacity) from the same donor to determine whether the epigenetic memory could influence iPS properties, preferentially generating cells similar of the donor somatic cell type. Until now, different approaches have been reported to generate MPs from iPS. So far, these methods present limitations such as low efficiency/reproducibility and usually involve cell sorting for enrichment or forced expression of skeletal master genes risking undesired genetic recombination. Recently, substantial interest is mounting regarding extracellular vesicles (EVs) and their involvement in many cellular processes, including myogenesis. We explored the possibility to use EVs as "physiological liposomes" enriched with myogenic factors to trigger skeletal myogenesis. To this end, during the second part of the study we developed a new transgenic-free approach to obtain transplantable MPs by means of defined factors and extracellular vesicles (EVs) secreted from differentiated mouse skeletal myoblasts. We established a novel, robust stepwise protocol by treating iPS with a WNT agonist, CHIR 99021 and myotubes-derived EVs. Thus, this method has two main advantages: (i) studying molecular mechanisms of myogenesis which is overpassed in case of genetic manipulation; (ii) muscle progenitors are not terminally differentiated, and therefore have a better repair potential following transplantation. One of the major hurdles of stem cell therapy for skeletal muscle regeneration is the massive death following transplantation. Biomaterials exhibit immune protection properties and would ensure an artificial microenvironment which permits them to interact with host cells and exert their therapeutic benefits. With the purpose of a better engraftment, we employed Poly (ethylene glycol) (PEG) -fibrinogen hydrogel (PF) as cell carrier for skeletal muscle regeneration. When transplanted in a αsarcoglycan knockout/severe combined immunodeficiency beige (α-SGKO/SCIDbg) mice, PF-embedded myogenic progenitor cells exhibited stable long-term engraftment and participated in muscle regeneration by fusing with existing muscle fibers. Importantly, no teratoma and no abnormal structure were detected in the muscles transplanted with MPs Finally, our finding and differentiation system provide an effective method that facilitates further utilization of iPS