Project description:STAC3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon STAC3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, STAC3 was initially expressed in myotomal adaxial cells and in fast muscle fibres post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibres, and decreased levels of the hedgehog receptor patched. The function of STAC3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. STAC3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of STAC3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton and the pro-myogenic factors IGFBP-5 and IGF2 were down regulated. RNAi-treated cells had suppressed AKT signalling and exogenous IGF2 was unable to rescue the phenotype, however, IGF/AKT signalling was not blocked. Overexpression of STAC3, which results in increased levels of IGFBP-5 mRNA, did not lead to increased differentiation. In synchronized cells, STAC3 mRNA was most abundant during the G1 phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and decreased proportion of cells in G1 phase when compared to controls, suggesting a role in the G1 phase checkpoint. These results identify STAC3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates. We identified STAC3 as being essential for myogenic differentiation. The goal of the microarray analysis was to identify genes that were up- or down-regulated in C2C12 cells when STAC3 expression was inhibited by interfering RNA (RNAi). The microarray compares the C2C12 mouse myogenic cell line between control samples to samples treated with an interfering RNA directed at STAC3. The mouse C2C12 cell line was used at two time points: day 0, which was 24 hours after adding the RNAi, with cells growing in growth media (non-differentiating), and day 1, which was 48 hours after the interfering RNAi was added, and 24 hours after switching to differentiation media. RNAi-treated cells were transfected with an RNAi directed against STAC3, and control cells were transfected with a high-GC content control RNAi. Total RNA was extracted from control and RNAi-treated samples at two time points, at day 0 (24 h after treating with RNAi, while samples were still in growth media), and at day 1 (cells grown in differentiation media for 24h, 48 h after RNAi treatment). At each time point, STAC3 RNAi-treated cells were compared to controls. For the array comparison, at day 0, triplicate biological samples + one technical replicate were used for the controls, and four biological samples were used for the RNAi-treated cells. At day 1, four biological samples and one technical replicate were used for controls and RNAi-treated cells. Technical replicate identifiers are appended by _r.

Project description:STAC3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon STAC3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, STAC3 was initially expressed in myotomal adaxial cells and in fast muscle fibres post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibres, and decreased levels of the hedgehog receptor patched. The function of STAC3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. STAC3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of STAC3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton and the pro-myogenic factors IGFBP-5 and IGF2 were down regulated. RNAi-treated cells had suppressed AKT signalling and exogenous IGF2 was unable to rescue the phenotype, however, IGF/AKT signalling was not blocked. Overexpression of STAC3, which results in increased levels of IGFBP-5 mRNA, did not lead to increased differentiation. In synchronized cells, STAC3 mRNA was most abundant during the G1 phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and decreased proportion of cells in G1 phase when compared to controls, suggesting a role in the G1 phase checkpoint. These results identify STAC3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates. We identified STAC3 as being essential for myogenic differentiation. The goal of the microarray analysis was to identify genes that were up- or down-regulated in C2C12 cells when STAC3 expression was inhibited by interfering RNA (RNAi). The microarray compares the C2C12 mouse myogenic cell line between control samples to samples treated with an interfering RNA directed at STAC3. The mouse C2C12 cell line was used at two time points: day 0, which was 24 hours after adding the RNAi, with cells growing in growth media (non-differentiating), and day 1, which was 48 hours after the interfering RNAi was added, and 24 hours after switching to differentiation media.

Project description:BACKGROUND:Understanding stem cell differentiation is essential for the future design of cell therapies. While retinoic acid (RA) is the most potent small molecule enhancer of skeletal myogenesis in stem cells, the stage and mechanism of its function has not yet been elucidated. Further, the intersection of RA with other signalling pathways that stimulate or inhibit myogenesis (such as Wnt and BMP4, respectively) is unknown. Thus, the purpose of this study is to examine the molecular mechanisms by which RA enhances skeletal myogenesis and interacts with Wnt and BMP4 signalling during P19 or mouse embryonic stem (ES) cell differentiation. RESULTS:Treatment of P19 or mouse ES cells with low levels of RA led to an enhancement of skeletal myogenesis by upregulating the expression of the mesodermal marker, Wnt3a, the skeletal muscle progenitor factors Pax3 and Meox1, and the myogenic regulatory factors (MRFs) MyoD and myogenin. By chromatin immunoprecipitation, RA receptors (RARs) bound directly to regulatory regions in the Wnt3a, Pax3, and Meox1 genes and RA activated a beta-catenin-responsive promoter in aggregated P19 cells. In the presence of a dominant negative beta-catenin/engrailed repressor fusion protein, RA could not bypass the inhibition of skeletal myogenesis nor upregulate Meox1 or MyoD. Thus, RA functions both upstream and downstream of Wnt signalling. In contrast, it functions downstream of BMP4, as it abrogates BMP4 inhibition of myogenesis and Meox1, Pax3, and MyoD expression. Furthermore, RA downregulated BMP4 expression and upregulated the BMP4 inhibitor, Tob1. Finally, RA inhibited cardiomyogenesis but not in the presence of BMP4. CONCLUSION:RA can enhance skeletal myogenesis in stem cells at the muscle specification/progenitor stage by activating RARs bound directly to mesoderm and skeletal muscle progenitor genes, activating beta-catenin function and inhibiting bone morphogenetic protein (BMP) signalling. Thus, a signalling pathway can function at multiple levels to positively regulate a developmental program and can function by abrogating inhibitory pathways. Finally, since RA enhances skeletal muscle progenitor formation, it will be a valuable tool for designing future stem cell therapies.

Project description:BackgroundDuchenne muscular dystrophy (DMD) is caused by mutations of the gene that encodes the protein dystrophin. A loss of dystrophin leads to severe and progressive muscle wasting in both skeletal and heart muscles. Human induced pluripotent stem cells (hiPSCs) and their derivatives offer important opportunities to treat a number of diseases. Here, we investigated whether givinostat (Givi), a histone deacetylase inhibitor, with muscle differentiation properties could reprogram hiPSCs into muscle progenitor cells (MPC) for DMD treatment.MethodsMPC were generated from hiPSCs by treatment with CHIR99021 and givinostat called Givi-MPC or with CHIR99021 and fibroblast growth factor as control-MPC. The proliferation and migration capacity were investigated by CCK-8, colony, and migration assays. Engraftment, pathological changes, and restoration of dystrophin were evaluated by in vivo transplantation of MPC. Conditioned medium from cultured MPC was collected and analyzed for extracellular vesicles (EVs).ResultsGivi-MPC exhibited superior proliferation and migration capacity compared to control-MPC. Givi-MPC produced less reactive oxygen species (ROS) after oxidative stress and insignificant expression of IL6 after TNF-α stimulation. Upon transplantation in cardiotoxin (CTX)-injured hind limb of Mdx/SCID mice, the Givi-MPC showed robust engraftment and restored dystrophin in the treated muscle than in those treated with control-MPC or human myoblasts. Givi-MPC significantly limited infiltration of inflammatory cells and reduced muscle necrosis and fibrosis. Additionally, Givi-MPC seeded the stem cell pool in the treated muscle. Moreover, EVs released from Givi-MPC were enriched in several miRNAs related to myoangiogenesis including miR-181a, miR-17, miR-210 and miR-107, and miR-19b compared with EVs from human myoblasts.ConclusionsIt is concluded that hiPSCs reprogrammed into MPC by givinostat possessing anti-oxidative, anti-inflammatory, and muscle gene-promoting properties effectively repaired injured muscle and restored dystrophin in the injured muscle.

Dataset Information

Inhibition of the Combinatorial Signaling of Transforming Growth Factor-Beta and NOTCH Promotes Myotube Formation of Human Pluripotent Stem Cell-Derived Skeletal Muscle Progenitor Cells.

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Inhibition of the Combinatorial Signaling of Transforming Growth Factor-Beta and NOTCH Promotes Myotube Formation of Human Pluripotent Stem Cell-Derived Skeletal Muscle Progenitor Cells.

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