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 ¼ 11) were labeled with fluorescent antibodies for Pax7 (SC transcriptional marker), laminin (basal lamina), and 4 0 ,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 AE 0.39) compared to TD children (8.77 AE 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. ß

Synthetic RNA-based genetic devices dynamically control a wide range of gene-regulatory processes across diverse cell types. However, the limited throughput of quantitative assays in mammalian cells has hindered fast iteration and interrogation of sequence space needed to identify new RNA devices. Here we report developing a quantitative, rapid and high-throughput mammalian cell-based RNA-Seq assay to efficiently engineer RNA devices. We identify new ribozyme-based RNA devices that respond to theophylline, hypoxanthine, cyclic-di-GMP, and folinic acid from libraries of ~22,700 sequences in total. The small molecule responsive devices exhibit low basal expression and high activation ratios, significantly expanding our toolset of highly functional ribozyme switches. The large datasets obtained further provide conserved sequence and structure motifs that may be used for rationally guided design. The RNA-Seq approach offers a generally applicable strategy for developing broad classes of RNA devices, thereby advancing the engineering of genetic devices for mammalian systems.

Introduction Muscles add sarcomeres in response to stretch, presumably to maintain optimal sarcomere length. Clinical evidence from patients with cerebral palsy (CP), 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 their capacity for sarcomere addition. Muscle architecture, morphology, and extra-cellular 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 extra-cellular matrix (ECM) changes, consistent with fibrosis. Discussion While a reduced SC population does not hinder serial sarcomere addition, SCs do play a role in muscle adaptation to chronic stretch that involves maintenance of both fiber cross-sectional area and ECM structure.

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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 (Pax7CreER/+; Rosa26DTA/+; 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 (r2 =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.