In normal muscle, CD45-Sca1-Mac1-CXCR4+1-integrin+ (CSM4B) cells (blue gate) are enriched for PAX7-expressing satellite cells with myogenic precursor function [11, 1], and CD45-Sca1+ cells (red gate) are enriched for non-myogenic, adipogenic precursors [10, 3, 31]

In normal muscle, CD45-Sca1-Mac1-CXCR4+1-integrin+ (CSM4B) cells (blue gate) are enriched for PAX7-expressing satellite cells with myogenic precursor function [11, 1], and CD45-Sca1+ cells (red gate) are enriched for non-myogenic, adipogenic precursors [10, 3, 31]. in freshly isolated muscle satellite cells enhanced terminal myogenic differentiation without stimulating proliferation. Our findings support the conclusion that SmoM2 tumors Ibotenic Acid represent an aberrant skeletal muscle state and demonstrate that, similar to normal muscle, myogenic tumors contain functionally distinct cell subsets, including cells lacking myogenic differentiation potential. Keywords: Skeletal muscle, differentiation, Hedgehog signaling, intratumoral cellular heterogeneity Introduction Adult striated muscle is composed of highly organized bundles of multinucleated myofibers and a variety of functionally heterogeneous mononuclear cells [1C3], including myogenic (muscle-forming) and non-myogenic elements such as fibroadipogenic precursors (FAPs) and immune/ inflammatory cells of hematopoietic lineage. Within the myogenic cell compartment, cytoplasmic filaments such as Desmin, Actin and Myosin mark terminal myogenic differentiation, whereas the transcription factor PAX7 identifies satellite cells within the heterogenous pool of myofiber-associated mononuclear cells [2]. Upon injury, satellite cells proliferate, differentiate and fuse to generate new myofibers in a process that is governed by sequential expression of a series of myogenic regulatory factors including MyoD and Myogenin [4, 5]. These myogenic regulatory factors (MRFs) are generally silent in mature, resting muscle. Skeletal muscle differentiation Ibotenic Acid features can be found in a number of neoplastic conditions, including rhabdomyosarcomas, a varied group of soft-tissue sarcomas, and rhabdomyomas, benign tumors of striated muscle. These conditions have previously been linked to activation of certain oncogenic pathways, including activating mutations in Hedgehog (Hh) pathway genes, detected in fusion-negative human rhabdomyosarcomas [6, 7] and fetal rhabdomyomas [8, 7]. These tumors exhibit both terminal muscle differentiation markers (e.g. Actin) and myogenic regulatory factors (e.g. MyoD), and they represent an abnormal state of muscle differentiation [8, 9]. This study sought to examine cellular heterogeneity in myogenic tumors. We demonstrate that tumors arising in mouse skeletal muscle following induction of hyperactive Hh signaling Ibotenic Acid [8, 9] recapitulate normal skeletal muscle cellular heterogeneity and contain an expanded pool of PAX7+, MyoD+ satellite-like cells. Material and methods Mice R26-SmoM2(+/?) and R26-SmoM2(+/+) (mixed genetic background including 129/Sv and Swiss Webster as main components) [9] and R26-SmoM2(+/?);CAGGS-CreER [9] were bred at the Joslin Diabetes Center Animal Facility. Throughout this manuscript, R26-SmoM2(+/?) or R26-SmoM2(+/+) skeletal muscle is referred to as control muscle, and R26-SmoM2(+/?);CAGGS-CreER skeletal muscle as SmoM2 muscle. C57BL6 mice were purchased from the Jackson Laboratory. Tamoxifen (Sigma, St Louis, MO) at a dose of 1 1 mg/40 g body weight was administered to R26-SmoM2(+/?);CAGGS-CreER intraperitoneally on postnatal day 10 (P10) to activate CreER-mediated recombination at transgene-encoded loxP sites. High rates of recombination in skeletal muscle were previously documented [9]. R26-SmoM2;CAGGS-CreER mice were monitored once weekly for the onset of soft-tissue tumors or other health problems, and they were sacrificed once they were ill. All animal experiments were approved by the Joslin Diabetes Center Institutional Animal Care and Use Committee. Histopathological evaluation of skeletal muscle and Rcan1 tumors Skeletal muscle and tumor tissue was dissected, fixed in 4% paraformaldehyde for 2 hours, and embedded in paraffin. Standard H&E stained sections were prepared. Staining for Myogenin (Dako, M3559, 1:100), MyoD1 (Dako, M3512, 1:50), Desmin (Dako, M0760, Ibotenic Acid 1:50), FABP4 (Cell Signaling, D25B3, 1 :200), CD45 (Abcam, ab10558, 1:4000) and PAX7 (DSHB, 1:5) was performed as previously described [2]. Muscle and tumor dissociation Upper extremity, lower extremity and pectoralis muscles from 4C8 week-old C57BL6/J wild-type, 4C9 week-old R26-SmoM2 mice and 3C9 week-old, tamoxifen-induced R26-SmoM2;CAGGS-CreER mice were harvested. Isolation of myofiber-associated cells was performed by two-step enzymatic digestion and mechanical dissociation as previously described [1]. Isolation of SmoM2 tumor cells was performed by one-step enzymatic digestion and mechanical dissociation as follows: Tumors were harvested, digested in DMEM + 0.2% collagenase type II (Invitrogen) + 0.05% dispase (Invitrogen) for 90 minutes at 37C in a shaking waterbath, triturated to disrupt the remaining tumor pieces and filtered through a 70m cell strainer. Red Ibotenic Acid blood cells were lysed from tumor cell preparations by 3 min incubation in 0.15M ammonium chloride, 0.01M potassium bicarbonate solution on ice. Fluorescence activated cell sorting (FACS) of myofiber-associated and tumor cells Phenotypically distinct muscle and tumor cell subsets were sorted according to protocols that were previously established to isolate functionally discrete subsets of myofiber-associated cells [10, 11, 1]. In brief, cells were.

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