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An indium-111-labelled peptide binds to myeloma cells, enabling SPECT imaging of myeloma cell accumulation in lungs after intravenous administration. This is the first report of a radiolabelled cell-membrane binding peptide for cell tracking.

Rhabdomyosarcoma is a rare childhood soft tissue cancer whose cells resemble poorly differentiated skeletal muscle, expressing myogenic proteins including MYOGENIN. Alveolar rhabdomyosarcoma (ARMS) accounts for ~40% of cases and is associated with a poorer prognosis than other rhabdomyosarcoma variants, especially if containing the chromosomal translocation generating the PAX3-FOXO1 hybrid transcription factor. Metastasis is commonly present at diagnosis, with a five-year survival rate of MYOGENIN promoter by deleting both the NF1 and MEF3 transcription factor binding motifs to produce a promoter that is highly active in ARMS cells. Our bespoke ARMS promoter driving HSV-TK efficiently killed ARMS cells in vitro, but not skeletal myoblasts. Using a xenograft mouse model, we also demonstrated that ARMS promoter-HSV-TK causes apoptosis of ARMS cells in vivo. Importantly, combining our suicide gene therapy with standard chemotherapy agents used in the treatment of rhabdomyosarcoma, reduced the effective drug dose, diminishing deleterious side effects/patient burden. This modified, highly ARMS-specific promoter could provide a new therapy option for this difficult-to-treat cancer.

Facioscapulohumeral muscular dystrophy (FSHD) is characterised by descending skeletal muscle weakness and wasting. FSHD is caused by mis-expression of the transcription factor DUX4, which is linked to oxidative stress, a condition especially detrimental to skeletal muscle with its high metabolic activity and energy demands. Oxidative damage characterises FSHD and recent work suggests metabolic dysfunction and perturbed hypoxia signalling as novel pathomechanisms. However, redox biology of FSHD remains poorly understood, and integrating the complex dynamics of DUX4-induced metabolic changes is lacking. Here we pinpoint the kinetic involvement of altered mitochondrial ROS metabolism and impaired mitochondrial function in aetiology of oxidative stress in FSHD. Transcriptomic analysis in FSHD muscle biopsies reveals strong enrichment for pathways involved in mitochondrial complex I assembly, nitrogen metabolism, oxidative stress response and hypoxia signalling. We found elevated mitochondrial ROS (mitoROS) levels correlate with increases in steady-state mitochondrial membrane potential in FSHD myogenic cells. DUX4 triggers mitochondrial membrane polarisation prior to oxidative stress generation and apoptosis through mitoROS, and affects mitochondrial health through lipid peroxidation. We identify complex I as the primary target for DUX4-induced mitochondrial dysfunction, with strong correlation between complex I-linked respiration and cellular oxygenation/hypoxia signalling activity in environmental hypoxia. Thus, FSHD myogenesis is uniquely susceptible to hypoxia-induced oxidative stress as a consequence of metabolic mis-adaptation. Importantly, mitochondria-targeted antioxidants rescue FSHD pathology more effectively than conventional antioxidants, highlighting the central involvement of disturbed mitochondrial ROS metabolism. This work provides a pathomechanistic model by which DUX4-induced changes in oxidative metabolism impair muscle function in FSHD, amplified when metabolic adaptation to varying O

Facioscapulohumeral muscular dystrophy (FSHD) is characterised by descending skeletal muscle weakness and wasting. FSHD is caused by mis-expression of the transcription factor DUX4, which is linked to oxidative stress, a condition especially detrimental to skeletal muscle with its high metabolic activity and energy demands. Oxidative damage characterises FSHD and recent work suggests metabolic dysfunction and perturbed hypoxia signalling as novel pathomechanisms. However, redox biology of FSHD remains poorly understood, and integrating the complex dynamics of DUX4-induced metabolic changes is lacking.Here we pinpoint the kinetic involvement of altered mitochondrial RONS metabolism and impaired mitochondrial function in aetiology of oxidative stress in FSHD. Transcriptomic analysis in FSHD muscle biopsies reveals strong enrichment for pathways involved in mitochondrial complex I assembly, nitrogen metabolism, oxidative stress response and hypoxia signalling. We found elevated ROS levels correlate with increases in steady-state mitochondrial membrane potential in FSHD myogenic cells. DUX4 triggers mitochondrial membrane polarisation prior to oxidative stress generation and apoptosis through mitochondrial ROS, and affects NO· bioavailability via mitochondrial peroxidation. We identify complex I as the primary target for DUX4-induced mitochondrial dysfunction, with strong correlation between complex I-linked respiration and cellular oxygenation/hypoxia signalling activity in environmental hypoxia. Thus, FSHD myogenesis is uniquely susceptible to hypoxia-induced oxidative stress as a consequence of metabolic mis-adaptation. Importantly, mitochondria-targeted antioxidants rescue FSHD pathology more effectively than conventional antioxidants, highlighting the central involvement of disturbed mitochondrial RONS metabolism. This work provides a pathomechanistic model by which DUX4-induced changes in oxidative metabolism impair muscle function in FSHD, amplified when metabolic adaptation to varying O2 tension is required.HighlightsTranscriptomics data from FSHD muscle indicates enrichment for disturbed mitochondrial pathwaysDisturbed RONS metabolism correlates with mitochondrial membrane polarisation and myotube hypotrophyDUX4-induced changes in mitochondrial function precede oxidative stress through mitoROS and affect hypoxia signalling via complex IFSHD is sensitive to environmental hypoxia, which increases ROS levels in FSHD myotubesHypotrophy in hypoxic FSHD myotubes is efficiently rescued with mitochondria-targeted antioxidantsGraphical abstract

Rhabdomyosarcoma is a rare childhood soft tissue cancer whose cells resemble poorly differentiated skeletal muscle, expressing myogenic proteins including MYOGENIN. Alveolar rhabdomyosarcoma (ARMS) accounts for ~40% of cases and is associated with a poorer prognosis than other rhabdomyosarcoma variants, especially if containing the chromosomal translocation generating the PAX3-FOXO1 hybrid transcription factor. Metastasis is commonly present at diagnosis, with a five-year survival rate of

Pepsin-solubilized atelocollagen can be used to form highly complex three-dimensional matrices for a broad spectrum of tissue engineering applications. Moreover, it has a long history as a favorable biomaterial in pharmaceutical and medical industries. So far, the main sources for these approaches are collagens from xenogenic sources. Yet, these nonhuman collagens carry a risk of provoking immune reactions in patients. Here we describe an effective method of isolating atelocollagen type 1/3 (COL1/3) from human placenta. By combining a single pepsin digestion step with tangential flow filtration and further precipitation steps, we could purify COL1/3 within only 4 days of processing. The resulting COL1/3 was biochemically characterized by determining residual DNA content, proving the absence of impurities by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis combined with total amino acid quantification, identifying the isolated collagen types by Western blot analysis, and analyzing the spontaneous formation of fibrous structures on freeze-drying via scanning electron microscopy. Finally, the cytocompatibility of the isolated collagen was demonstrated in two dimensional using primary rat hepatocytes and in three dimensional by a sprouting assay of human umbilical vein endothelial cell. The isolation method described not only fulfills demands for cost-efficient bioengineering using a human waste material but also potentially increases overall safety for patients by use of homologous products.

Objectives: DISHEVELLED, EGL‐10, PLECKSTRIN (DEP) domain‐containing 1B (DEPDC1B) promotes dismantling of focal adhesions and coordinates detachment events during cell cycle progression. DEPDC1B is overexpressed in several cancers with expression inversely correlated with patient survival. Here, we analysed the role of DEPDC1B in the regulation of murine and human skeletal myogenesis.Materials and methods: Expression dynamics of DEPDC1B were examined in murine and human myoblasts and rhabdomyosarcoma cells in vitro by RT‐qPCR and/or immunolabelling. DEPDC1B function was mainly tested via siRNA‐mediated gene knockdown.Results: DEPDC1B was expressed in proliferating murine and human myoblasts, with expression then decreasing markedly during myogenic differentiation. SiRNA‐mediated knockdown of DEPDC1B reduced myoblast proliferation and induced entry into myogenic differentiation, with deregulation of key cell cycle regulators (cyclins, CDK, CDKi). DEPDC1B and β‐catenin co‐knockdown was unable to rescue proliferation in myoblasts, suggesting that DEPDC1B functions independently of canonical WNT signalling during myogenesis. DEPDC1B can also suppress RHOA activity in some cell types, but DEPDC1B and RHOA co‐knockdown actually had an additive effect by both further reducing proliferation and enhancing myogenic differentiation. DEPDC1B was expressed in human Rh30 rhabdomyosarcoma cells, where DEPDC1B or RHOA knockdown promoted myogenic differentiation, but without influencing proliferation.Conclusion: DEPDC1B plays a central role in myoblasts by driving proliferation and preventing precocious myogenic differentiation during skeletal myogenesis in both mouse and human.

The generation of functional biomimetic skeletal muscle constructs is still one of the fundamental challenges in skeletal muscle tissue engineering. With the notion that structure strongly dictates functional capabilities, a myriad of cell types, scaffold materials and stimulation strategies have been combined. To further optimize muscle engineered constructs, we have developed a novel bioreactor system (MagneTissue) for rapid engineering of skeletal muscle-like constructs with the aim to resemble native muscle in terms of structure, gene expression profile and maturity. Myoblasts embedded in fibrin, a natural hydrogel that serves as extracellular matrix, are subjected to mechanical stimulation via magnetic force transmission. We identify static mechanical strain as a trigger for cellular alignment concomitant with the orientation of the scaffold into highly organized fibrin fibrils. This ultimately yields myotubes with a more mature phenotype in terms of sarcomeric patterning, diameter and length. On the molecular level, a faster progression of the myogenic gene expression program is evident as myogenic determination markers MyoD and Myogenin as well as the Ca2+ dependent contractile structural marker TnnT1 are significantly upregulated when strain is applied. The major advantage of the MagneTissue bioreactor system is that the generated tension is not exclusively relying on the strain generated by the cells themselves in response to scaffold anchoring but its ability to subject the constructs to individually adjustable strain protocols. In future work, this will allow applying mechanical stimulation with different strain regimes in the maturation process of tissue engineered constructs and elucidating the role of mechanotransduction in myogenesis. Statement of Significance Mechanical stimulation of tissue engineered skeletal muscle constructs is a promising approach to increase tissue functionality. We have developed a novel bioreactor-based 3D culture system, giving the user the possibility to apply different strain regimes like static, cyclic or ramp strain to myogenic precursor cells embedded in a fibrin scaffold. Application of static mechanical strain leads to alignment of fibrin fibrils along the axis of strain and concomitantly to highly aligned myotube formation. Additionally, the pattern of myogenic gene expression follows the temporal progression observed in vivo with a more thorough induction of the myogenic program when static strain is applied. Ultimately, the strain protocol used in this study results in a higher degree of muscle maturity demonstrated by enhanced sarcomeric patterning and increased myotube diameter and length. The introduced bioreactor system enables new possibilities in muscle tissue engineering as longer cultivation periods and different strain applications will yield tissue engineered muscle-like constructs with improved characteristics in regard to functionality and biomimicry.