Engineering a 3D in vitro model of human skeletal muscle at the single fiber scale

The reproduction of reliable in vitro models of human skeletal muscle is made harder by the intrinsic 3D structural complexity of this tissue. Here we coupled engineered hydrogel with 3D structural cues and specific mechanical properties to derive human 3D muscle constructs ("myobundles") at the scale of single fibers, by using primary myoblasts or myoblasts derived from embryonic stem cells. To this aim, cell culture was performed in confined, laminin-coated micrometric channels obtained inside a 3D hydrogel characterized by the optimal stiffness for skeletal muscle myogenesis. Primary myoblasts cultured in our 3D culture system were able to undergo myotube differentiation and maturation, as demonstrated by the proper expression and localization of key components of the sarcomere and sarcolemma. Such approach allowed the generation of human myobundles of ~10 mm in length and ~120 \u3bcm in diameter, showing spontaneous contraction 7 days after cell seeding. Transcriptome analyses showed higher similarity between 3D myobundles and skeletal signature, compared to that found between 2D myotubes and skeletal muscle, mainly resulting from expression in 3D myobundles of categories of genes involved in skeletal muscle maturation, including extracellular matrix organization. Moreover, imaging analyses confirmed that structured 3D culture system was conducive to differentiation/maturation also when using myoblasts derived from embryonic stem cells. In conclusion, our structured 3D model is a promising tool for modelling human skeletal muscle in healthy and diseases conditions

Micro-engineered skeletal and cardiac muscle for Duchenne muscular dystrophy in vitro models

Duchenne muscular dystrophy (DMD) is the most common and severe genetic neuromuscular disorder affecting both skeletal and cardiac muscle functionality. More than twenty years have passed since the identification of the dystrophin gene, which mutations cause the disease. Many progresses have been made in understanding the pathogenesis and different experimental strategies has been tested both in vitro, on bench-top cell cultures, and in vivo, on different animal models. So far, despite some promising outcomes coming from recent clinical trials, this has not resulted in an effective and definitive cure significantly altering the relentless progression of this disease, which has still a 100% mortality rate. In this context, the aim of this PhD thesis is the development of micro-engineered human skeletal and cardiac muscles representing in vitro models of DMD patient tissues useful for testing therapeutic approaches aimed at restoring a proper dystrophin expression. The strategy applied for the obtainment of such human in vitro models rely on the application of micro-scale technologies for reproducing in vitro the main physiological cues that guide differentiation and allow functionality of skeletal and cardiac muscles in vivo. In particular, the mechanical properties of the cell micro-environment and the topologic organization over the cell culture were optimized for both skeletal and cardiac muscle. Such micro-scale technologies were coupled with an appropriate human cell source. Human primary myoblasts from biopsies of DMD patient were used for skeletal muscle engineering, while DMD patient-specific cardiomyocytes were differentiated from human pluripotent stem (hiPS) cells for modeling the cardiac muscle. Both the obtained DMD in vitro models were validated for testing the ability of different therapeutic approaches in restoring dystrophin expression. In particular, three different myogenic cell types were tested on the engineered DMD skeletal muscle, while dystrophin expression restoration by a human artificial chromosome carrying the full-length genomic dystrophin sequence was tested on hiPS cells-derived cardiomyocytes. In these perspectives, the developed human in vitro models can represent a useful platform for performing preliminary or pre-clinical tests of different therapeutic strategy for DMD. In addition, they can be used as complementary tool in a clinical trial, for test different batches of cells before using them on patients.La distrofia muscolare di Duchenne è una delle più frequenti e severe patologie genetiche neuromuscolari che affliggono la funzionalità del muscolo scheletrico e cardiaco. Il gene codificante la distrofina, proteina la cui mutazione è alla base della patologia, è stato scoperto più di vent’anni fa. Da allora, notevoli progressi sono stati compiuti nella comprensione della patogenesi di questa malattia e diverse strategie sperimentali volte al suo trattamento sono state testate, sia in vitro su convenzionali colture cellulari che in vivo su diversi animali modello. Tuttavia, eccezione fatta per alcuni promettenti risultati recentemente ottenuti in trials clinici, ad oggi non vi è ancora una cura efficace e definitiva in grado di alterare o rallentare la progressione di questa patologia, il cui tasso di mortalità è pari al 100%. In tale contesto, lo scopo di questa tesi di dottorato è quello di sviluppare dei modelli in vitro micro-ingegnerizzati di muscolo scheletrico e cardiaco umano, che siano rappresentativi dei tessuti distrofici e dunque utili per testare approcci terapeutici volti al ripristino dell’espressione di distrofina. La strategia applicata per l’ottenimento di tali modelli si basa sull’applicazione di tecnologie su microscala per riprodurre in vitro i principali stimoli che guidano il differenziamento e consentono la funzionalità del muscolo scheletrico e cardiaco in vivo. In particolare, le proprietà meccaniche del micro-ambiente e l’organizzazione topologica della coltura cellulare sono stati ottimizzati sia per il muscolo scheletrico che cardiaco. Tali tecnologie su micro-scala sono state accoppiate con un’appropriata fonte cellulare umana. Per l’ingegnerizzazione del muscolo scheletrico sono stati utilizzati mioblasti umani primari derivanti da biopsie di pazienti DMD mentre, per la modellazione del muscolo cardiaco, cellule umane pluripotenti indotte (iPS) sono state differenziate in cardiomiociti paziente-specifici. Entrambi i modelli in vitro di muscolo distrofico ottenuti sono stati validati testando l’abilità di diversi approcci terapeutici nel ripristinarne l’espressione di distrofina. In particolare, tre diversi tipi cellulari miogenici sono stati testati nel muscolo scheletrico distrofico ingegnerizzato. Inoltre, nei cardiomiociti distrofici derivanti da cellule iPS è stato testato il ripristino dell’espressione di distrofina per mezzo di un cromosoma artificiale portante la sua completa sequenza genomica. Da tali risultati emerge come i modelli umani in vitro sviluppati in questo lavoro possano rappresentare un’utile piattaforma su cui effettuare test pre-clinici preliminari di diverse strategie terapeutiche. Inoltre, essi posso potenzialmente essere utilizzati come strumento complementare durante i trials clinici, per testare, ad esempio, diverse preparazioni di cellule destinate al paziente

Substrate and mechanotransduction influence SERCA2a localization in human pluripotent stem cell-derived cardiomyocytes affecting functional performance

Physical cues are major determinants of cellular phenotype and evoke physiological and pathological responses on cell structure and function. Cellular models aim to recapitulate basic functional features of their in vivo counterparts or tissues in order to be of use in in vitro disease modeling or drug screening and testing. Understanding how culture systems affect in vitro development of human pluripotent stem cell (hPSC)-derivatives allows optimization of cellular human models and gives insight in the processes involved in their structural organization and function. In this work, we show involvement of the mechanotransduction pathway RhoA/ROCK in the structural reorganization of hPSC-derived cardiomyocytes after adhesion plating. These structural changes have a major impact on the intracellular localization of SERCA2 pumps and concurrent improvement in calcium cycling. The process is triggered by cell interaction with the culture substrate, which mechanical cues drive sarcomeric alignment and SERCA2a spreading and relocalization from a perinuclear to a whole-cell distribution. This structural reorganization is mediated by the mechanical properties of the substrate, as shown by the process failure in hPSC-CMs cultured on soft 4 kPa hydrogels as opposed to physiologically stiff 16 kPa hydrogels and glass. Finally, pharmacological inhibition of Rho-associated protein kinase (ROCK) by different compounds identifies this specific signaling pathway as a major player in SERCA2 localization and the associated improvement in hPSC-CMs calcium handling ability in vitro

ENGINEERING A 3D IN VITRO MODEL OF HUMAN SKELETAL MUSCLE

Myofibers, the basic structural elements of skeletal muscle tissue, are formed and regenerated after injury in a unique series of events that include myoblasts adhesion, fusion and differentiation. In this process a key role is played by morphological, mechanical and biochemical stimuli provided by the extracellular environment in vivo. Traditional in vitro two-dimensional (2D) cell culture systems have been very useful to elucidate early steps of myogenesis. However, cells cultured on flat substrates differ considerably in their morphology, cell-cell/cell-matrix interaction, and differentiation from those in the physiological threedimensional (3D) environments. The aim of this work was to engineer three-dimensional (3D) human skeletal myofibers in vitro for: i) studying human myogenesis in an in vivo-like physiological microenvironment, ii) developing 3D implantable myofibers for repairing muscle defects. To achieve myoblasts spatial organization and alignment, we designed a soft hydrogel (HY) scaffold with 3D parallel micro-channels (80-160 \u3bcm in diameter, 10-15 mm long) functionalized with Matrigel. The HY ensures mass transport of metabolite and cytokines required for the proper myoblasts growth and differentiation. HY chemical composition was optimized in order to obtain a soft scaffold surrounding myobasts and myotubes, with mechanical properties (elastic modulus, E) similar to those of the physiological microenvironment of muscle in vivo (E 4812\ub14kPa). Human myoblasts (1\uf73x104 cells/channel) were injected into the micro-channels and cultured for up to 10 days. The composition of HY was optimized based on the final application: poly-acrylamide was used for in vitro studies, while hyaluronic acid for in vivo experiments. The developed HY were biocompatible and maintained the expression of myogenic markers such as desmin. After 10 days of culture, tightly packed human myotubes bundles have been obtained, expressing the differentiation markers myosin heavy chain, \u3b1-actinin and dystrophin. It is worth to underline that we observed spontaneous contractions of human myotubes bundles. Further to be three-dimensional, thanks to their relevant dimensions (up to 15 mm in length) and their compact and elastic nature, myotubes bundles could be easily manipulated for surgical implantation. GFP+ve muscle precursors cells were cultured into the channels and implanted in the tibialis anterioris of syngenic wilde type mice. After two weeks, the HY scaffold was completed degraded, without forming fibrous tissue, and implanted cells migrated from the implantation site and gave rise to newly formed myofibers: GFP+ve with central nuclei. Taken together, the obtained results showed that the 3D HY scaffold surrounding myoblasts simulates in vitro the mechanical and biochemical properties of the physiological cell microenvironment, allowing the formation of human differentiated and contracting myotubes bundles. On the other hand, in vivo studies showed an optimal degradability of the scaffold and the formation of new myofibers integrated within the host tissue

Micropatterning topology on soft substrates affects myoblasts proliferation and differentiation

Micropatterning techniques and substrate engineering are becoming useful tools to investigate several aspects of cell cell interaction biology. In this work, we rationally study how different micropatterning geometries can affect myoblast behavior in the early stage of in vitro myogenesis. Soft hydrogels with physiological elastic modulus (E = 15 kPa) were micropatterned in parallel lanes (100, 300, and 500 mu m width) resulting in different local and global myoblast densities. Proliferation and differentiation into multinucleated myotubes were evaluated for murine and human myoblasts. Wider lanes showed a decrease in murine myoblast proliferation: (69 +/- 8)% in 100 mu m wide lanes compared to (39 +/- 7)% in 500 mu m lanes. Conversely, fusion index increased in wider lanes: from (46 +/- 7)% to (66 +/- 7)% for murine myoblasts, and from (15 +/- 3)% to (36 +/- 2)% for human primary myoblasts, using a patterning width of 100 and 500 mu m, respectively. These results are consistent with both computational modeling data and conditioned medium experiments, which demonstrated that wider lanes favor the accumulation of endogenous secreted factors. Interestingly, human primary myoblast proliferation is not affected by patterning width, which may be because the high serum content of their culture medium overrides the effect of secreted factors. These data highlight the role of micropatterning in shaping the cellular niche through secreted factor accumulation, and are of paramount importance in rationally understanding myogenesis in vitro for the correct design of in vitro skeletal muscle models