Micropatterning topology on soft substrates affects myoblast 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 µm 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 µm wide lanes compared to (39 ± 7)% in 500 µm 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 µm, 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.

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 µm 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.

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 4kPa hydrogels as opposed to physiologically stiff 16kPa 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.

Skeletal Muscle Differentiation on a Chip Shows Human Donor Mesoangioblasts' Efficiency in Restoring Dystrophin in a Duchenne Muscular Dystrophy Model.

: Restoration of the protein dystrophin on muscle membrane is the goal of many research lines aimed at curing Duchenne muscular dystrophy (DMD). Results of ongoing preclinical and clinical trials suggest that partial restoration of dystrophin might be sufficient to significantly reduce muscle damage. Different myogenic progenitors are candidates for cell therapy of muscular dystrophies, but only satellite cells and pericytes have already entered clinical experimentation. This study aimed to provide in vitro quantitative evidence of the ability of mesoangioblasts to restore dystrophin, in terms of protein accumulation and distribution, within myotubes derived from DMD patients, using a microengineered model. We designed an ad hoc experimental strategy to miniaturize on a chip the standard process of muscle regeneration independent of variables such as inflammation and fibrosis. It is based on the coculture, at different ratios, of human dystrophin-positive myogenic progenitors and dystrophin-negative myoblasts in a substrate with muscle-like physiological stiffness and cell micropatterns. Results showed that both healthy myoblasts and mesoangioblasts restored dystrophin expression in DMD myotubes. However, mesoangioblasts showed unexpected efficiency with respect to myoblasts in dystrophin production in terms of the amount of protein produced (40% vs. 15%) and length of the dystrophin membrane domain (210-240 µm vs. 40-70 µm). These results show that our microscaled in vitro model of human DMD skeletal muscle validated previous in vivo preclinical work and may be used to predict efficacy of new methods aimed at enhancing dystrophin accumulation and distribution before they are tested in vivo, reducing time, costs, and variability of clinical experimentation. SIGNIFICANCE: This study aimed to provide in vitro quantitative evidence of the ability of human mesoangioblasts to restore dystrophin, in terms of protein accumulation and distribution, within myotubes derived from patients with Duchenne muscular dystrophy (DMD), using a microengineered model. An ad hoc experimental strategy was designed to miniaturize on a chip the standard process of muscle regeneration independent of variables such as inflammation and fibrosis. This microscaled in vitro model, which validated previous in vivo preclinical work, revealed that mesoangioblasts showed unexpected efficiency as compared with myoblasts in dystrophin production. Consequently, this model may be used to predict efficacy of new drugs or therapies aimed at enhancing dystrophin accumulation and distribution before they are tested in vivo.

Complete restoration of multiple dystrophin isoforms in genetically corrected Duchenne muscular dystrophy patient-derived cardiomyocytes.

Duchenne muscular dystrophy (DMD)-associated cardiac diseases are emerging as a major cause of morbidity and mortality in DMD patients, and many therapies for treatment of skeletal muscle failed to improve cardiac function. The reprogramming of patients' somatic cells into pluripotent stem cells, combined with technologies for correcting the genetic defect, possesses great potential for the development of new treatments for genetic diseases. In this study, we obtained human cardiomyocytes from DMD patient-derived, induced pluripotent stem cells genetically corrected with a human artificial chromosome carrying the whole dystrophin genomic sequence. Stimulation by cytokines was combined with cell culturing on hydrogel with physiological stiffness, allowing an adhesion-dependent maturation and a proper dystrophin expression. The obtained cardiomyocytes showed remarkable sarcomeric organization of cardiac troponin T and α-actinin, expressed cardiac-specific markers, and displayed electrically induced calcium transients lasting less than 1 second. We demonstrated that the human artificial chromosome carrying the whole dystrophin genomic sequence is stably maintained throughout the cardiac differentiation process and that multiple promoters of the dystrophin gene are properly activated, driving expression of different isoforms. These dystrophic cardiomyocytes can be a valuable source for in vitro modeling of DMD-associated cardiac disease. Furthermore, the derivation of genetically corrected, patient-specific cardiomyocytes represents a step toward the development of innovative cell and gene therapy approaches for DMD.

The transfer of human artificial chromosomes via cryopreserved microcells.

Microcell-mediated chromosome transfer (MMCT) technology enables a single and intact mammalian chromosome or megabase-sized chromosome fragments to be transferred from donor to recipient cells. The conventional MMCT method is performed immediately after the purification of microcells. The timing of the isolation of microcells and the preparation of recipient cells is very important. Thus, ready-made microcells can improve and simplify the process of MMCT. Here, we established a cryopreservation method to store microcells at -80 °C, and compared these cells with conventionally- (immediately-) prepared cells with respect to the efficiency of MMCT and the stability of a human artificial chromosome (HAC) transferred to human HT1080 cells. The HAC transfer in microcell hybrids was confirmed by FISH analysis. There was no significant difference between the two methods regarding chromosome transfer efficiency and the retention rate of HAC. Thus, cryopreservation of ready-to-use microcells provides an improved and simplified protocol for MMCT.

Micro-arrayed human embryonic stem cells-derived cardiomyocytes for in vitro functional assay.

INTRODUCTION: The heart is one of the least regenerative organs in the body and any major insult can result in a significant loss of heart cells. The development of an in vitro-based cardiac tissue could be of paramount importance for many aspects of the cardiology research. In this context, we developed an in vitro assay based on human cardiomyocytes (hCMs) and ad hoc micro-technologies, suitable for several applications: from pharmacological analysis to physio-phatological studies on transplantable hCMs. We focused on the development of an assay able to analyze not only hCMs viability, but also their functionality. METHODS: hCMs were cultured onto a poly-acrylamide hydrogel with tunable tissue-like mechanical properties and organized through micropatterning in a 20×20 array. Arrayed hCMs were characterized by immunofluorescence, GAP-FRAP analyses and live and dead assay. Their functionality was evaluated monitoring the excitation-contraction coupling. RESULTS: Micropatterned hCMs maintained the expression of the major cardiac markers (cTnT, cTnI, Cx43, Nkx2.5, α-actinin) and functional properties. The spontaneous contraction frequency was (0.83±0.2) Hz, while exogenous electrical stimulation lead to an increase up to 2 Hz. As proof of concept that our device can be used for screening the effects of pathological conditions, hCMs were exposed to increasing levels of H(2)O(2). Remarkably, hCMs viability was not compromised with exposure to 0.1 mM H(2)O(2), but hCMs contractility was dramatically suppressed. As proof of concept, we also developed a microfluidic platform to selectively treat areas of the cell array, in the perspective of performing multi-parametric assay. CONCLUSIONS: Such system could be a useful tool for testing the effects of multiple conditions on an in vitro cell model representative of human heart physiology, thus potentially helping the processes of therapy and drug development.

Soft substrates drive optimal differentiation of human healthy and dystrophic myotubes.

The in vitro development of human myotubes carrying genetic diseases, such as Duchenne Muscular Dystrophy, will open new perspectives in the identification of innovative therapeutic strategies. Through the proper design of the substrate, we guided the differentiation of human healthy and dystrophic myoblasts into myotubes exhibiting marked functional differentiation and highly defined sarcomeric organization. A thin film of photo cross-linkable elastic poly-acrylamide hydrogel with physiological-like and tunable mechanical properties (elastic moduli, E: 12, 15, 18 and 21 kPa) was used as substrate. The functionalization of its surface by micro-patterning in parallel lanes (75 microm wide, 100 microm spaced) of three adhesion proteins (laminin, fibronectin and matrigel) was meant to maximize human myoblasts fusion. Myotubes formed onto the hydrogel showed a remarkable sarcomere formation, with the highest percentage (60.0% +/- 3.8) of myotubes exhibiting sarcomeric organization, of myosin heavy chain II and alpha-actinin, after 7 days of culture onto an elastic (15 kPa) hydrogel and a matrigel patterning. In addition, healthy myotubes cultured in these conditions showed a significant membrane-localized dystrophin expression. In this study, the culture substrate has been adapted to human myoblasts differentiation, through an easy and rapid methodology, and has led to the development of in vitro human functional skeletal muscle myotubes useful for clinical purposes and in vitro physiological study, where to carry out a broad range of studies on human muscle physiopathology.