Modulating myoblast differentiation with RNA-based controllers.

Tunable genetic controllers play a critical role in the engineering of biological systems that respond to environmental and cellular signals. RNA devices, a class of engineered RNA-based controllers, enable tunable gene expression control of target genes in response to molecular effectors. RNA devices have been demonstrated in a number of systems showing proof-of-concept of applying ligand-responsive control over therapeutic activities, including regulation of cell fate decisions such as T cell proliferation and apoptosis. Here, we describe the application of a theophylline-responsive RNA device in a muscle progenitor cell system to control myogenic differentiation. Ribozyme-based RNA switches responsive to theophylline control fluorescent reporter expression in C2C12 myoblasts in a ligand dependent manner. HRAS and JAK1, both anti-differentiation proteins, were incorporated into RNA devices. Finally, we demonstrate that the regulation of HRAS expression via theophylline-responsive RNA devices results in the modulation of myoblast differentiation in a theophylline-dependent manner. Our work highlights the potential for RNA devices to exert drug-responsive, tunable control over cell fate decisions with applications in stem cell therapy and basic stem cell biology research.

Engineering synthetic RNA devices for cell control.

The versatility of RNA in sensing and interacting with small molecules, proteins and other nucleic acids while encoding genetic instructions for protein translation makes it a powerful substrate for engineering biological systems. RNA devices integrate cellular information sensing, processing and actuation of specific signals into defined functions and have yielded programmable biological systems and novel therapeutics of increasing sophistication. However, challenges centred on expanding the range of analytes that can be sensed and adding new mechanisms of action have hindered the full realization of the field's promise. Here, we describe recent advances that address these limitations and point to a significant maturation of synthetic RNA-based devices.

Stretch-induced satellite cell deformation incontracturedmuscles in children with cerebral palsy.

Satellite cells (SCs) are quiescent, adult skeletal muscle stem cells responsible for postnatal muscle growth and repair. Children with cerebral palsy (CP) have muscle contractures with reduced SC abundance, extracellular matrix abnormalities and reduced serial sarcomere number resulting in greatly increased in vivo sarcomere length, perhaps due to impaired sarcomere addition, compared to children with typical development (TD). Stretch is a strong activator of SCs that leads to addition of sarcomeres during bone-muscle growth. Mechanical loading and subsequent deformation of intracellular structures can lead to activation and proliferation, perhaps by cytoskeletal transmissions of extracellular mechanical signals to the nuclei. The primary aim of this study was to determine the effect of ex vivo stretch-induced sarcomere length change on SC deformation in children with CP and TD. Muscle biopsies were obtained from twelve children (7 CP, 5 TD) during surgery. Fiber bundles were labeled with fluorescent antibodies for Pax7 (SC), DRAQ5 (nuclei), and alpha-actinin (sarcomere protein). Fibers were stretched using a custom jig and imaged using confocal microscopy. SC nuclear length, height and aspect ratio underwent increased deformation with increasing sarcomere length (p < 0.05) in both groups. Slopes of association for SC nuclear length, aspect ratio and sarcomere lengths were similar between CP and TD. Our results indicate that SC in children with CP undergo similar deformation as TD across sarcomere lengths.

Does a Reduced Number of Muscle Stem Cells Impair the Addition of Sarcomeres and Recovery from a Skeletal Muscle Contracture? A Transgenic Mouse Model.

BACKGROUND: Children with cerebral palsy have impaired muscle growth and muscular contractures that limit their ROM. Contractures have a decreased number of serial sarcomeres and overstretched lengths, suggesting an association with a reduced ability to add the serial sarcomeres required for normal postnatal growth. Contractures also show a markedly reduced number of satellite cells-the muscle stem cells that are indispensable for postnatal muscle growth, repair, and regeneration. The potential role of the reduced number of muscle stem cells in impaired sarcomere addition leading to contractures must be evaluated. QUESTIONS/PURPOSES: (1) Does a reduced satellite cell number impair the addition of serial sarcomeres during recovery from an immobilization-induced contracture? (2) Is the severity of contracture due to the decreased number of serial sarcomeres or increased collagen content? METHODS: The hindlimbs of satellite cell-specific Cre-inducible mice (Pax7; Rosa26; n = 10) were maintained in plantarflexion with plaster casts for 2 weeks so that the soleus was chronically shortened and the number of its serial sarcomeres was reduced by approximately 20%. Subsequently, mice were treated with either tamoxifen to reduce the number of satellite cells or a vehicle (an injection and handling control). The transgenic mouse model with satellite cell ablation combined with a casting model to reduce serial sarcomere number recreates two features observed in muscular contractures in children with cerebral palsy. After 30 days, the casts were removed, the mice ankles were in plantarflexion, and the mice's ability to recover its ankle ROM by cage remobilization for 30 days were evaluated. We quantified the number of serial sarcomeres, myofiber area, and collagen content of the soleus muscle as well as maximal ankle dorsiflexion at the end of the recovery period. RESULTS: Mice with reduced satellite cell numbers did not regain normal ankle ROM in dorsiflexion; that is, the muscles remained in plantarflexion contracture (-16° ± 13° versus 31° ± 39° for the control group, -47 [95% confidence interval -89 to -5]; p = 0.03). Serial sarcomere number of the soleus was lower on the casted side than the contralateral side of the mice with a reduced number of satellite cells (2214 ± 333 versus 2543 ± 206, -329 [95% CI -650 to -9]; p = 0.04) but not different in the control group (2644 ± 194 versus 2729 ± 249, -85 [95% CI -406 to 236]; p = 0.97). The degree of contracture was strongly associated with the number of sarcomeres and myofiber area (r =0.80; P < 0.01) rather than collagen content. No differences were seen between groups in terms of collagen content and the fraction of muscle area. CONCLUSIONS: We found that a reduced number of muscle stem cells in a transgenic mouse model impaired the muscle's ability to add sarcomeres in series and thus to recover from an immobilization-induced contracture. CLINICAL RELEVANCE: The results of our study in transgenic mouse muscle suggests there may be a mechanistic relationship between a reduced number of satellite cells and a reduced number of serial sarcomeres. Contracture development, secondary to impaired sarcomere addition in muscles in children with cerebral palsy may be due to a reduced number of muscle stem cells.

Reduced skeletal muscle satellite cell number alters muscle morphology after chronic stretch but allows limited serial sarcomere addition.

INTRODUCTION: Muscles add sarcomeres in response to stretch, presumably to maintain optimal sarcomere length. Clinical evidence from patients with cerebral palsy, who have both decreased serial sarcomere number and reduced satellite cells (SCs), suggests a hypothesis that SCs may be involved in sarcomere addition. METHODS: A transgenic Pax7-DTA mouse model underwent conditional SC depletion, and their soleii were then stretch-immobilized to assess the capacity for sarcomere addition. Muscle architecture, morphology, and extracellular matrix (ECM) changes were also evaluated. RESULTS: Mice in the SC-reduced group achieved normal serial sarcomere addition in response to stretch. However, muscle fiber cross-sectional area was significantly smaller and was associated with hypertrophic ECM changes, consistent with fibrosis. CONCLUSIONS: While a reduced SC population does not hinder serial sarcomere addition, SCs play a role in muscle adaptation to chronic stretch that involves maintenance of both fiber cross-sectional area and ECM structure. Muscle Nerve 55: 384-392, 2017.

Reduced satellite cell number in situ in muscular contractures from children with cerebral palsy.

Satellite cells (SC) are quiescent adult muscle stem cells critical for postnatal development. Children with cerebral palsy have impaired muscular growth and develop contractures. While flow cytometry previously demonstrated a reduced SC population, extracellular matrix abnormalities may influence the cell isolation methods used, systematically isolating fewer cells from CP muscle and creating a biased result. Consequently, the purpose of this study was to use immunohistochemistry on serial muscle sections to quantify SC in situ. Serial cross-sections from human gracilis muscle biopsies (n = 11) were labeled with fluorescent antibodies for Pax7 (SC transcriptional marker), laminin (basal lamina), and 4',6-diamidino-2-phenylindole (nuclei). Fluorescence microscopy under high magnification was used to identify SC based on labeling and location. Mean SC/100 myofibers was reduced by ∼70% (p < 0.001) in children with CP (2.89 ± 0.39) compared to TD children (8.77 ± 0.79). Furthermore, SC distribution across fields was different (p < 0.05) with increased percentage of SC in fields being solitary cells (p < 0.01) in children with CP. Quantification of SC number in situ, without any other tissue manipulation confirms children with spastic CP have a reduced number. This stem cell loss may, in part, explain impaired muscle growth and apparent decreased responsiveness of CP muscle to exercise.