Project description:Background: Pleuropulmonary blastoma (PPB) is a rare lung malignancy in children. Here, we classified PPB subpopulations with Single-cell RNA sequencing. Methods: This study included 10,007 single cells from a girl with PPB. After choosing malignant tumor cells with an inferring copy number variation, we used non-negative matrix factorization and Seurat analysis to cluster the cells and divided the subgroups by their differentially expressed genes and Gene Ontology enrichment analysis. Additionally, pseudotime trajectory analysis of PPB was conducted with Monocle. Results: Tumor cells were divided into muscle (DEShiTNNT1hiTNNI1hi) and cartilage (TWIST1hiHTRA1hiBMP4hi). In cartilage, SOX9hiPAX1hiPAX9hi prechondrocytes, OGNhiMGPhi chondrocytes and DKK2hi chondrocytes were defined. In muscle lineage, PAX7hiMYF5hi satellite myogenic cells and MYOGhiMEF2ChiMYOD1hi myocytes were defined. And a new undefined FABP7hi satellite cells subset was found to function like another known ITM2Ahi satellite cells subset. As the promoters of muscle cell development, both were responsible for establishing membrane protein and proliferating. Conclusions: Our results roughly defined muscle heterogeneity in PPB and found activated and proliferating FABP7 satellite myogenic cells. And these satellite cells might be activated by microRNA regulation induced by the mutation of PPB classic mutation gene DICER1.
Project description:The contribution of non-resident, non-satellite myogenic progenitors to postnatal muscle homeostasis and repair is controversial. Precursor cells with the capacity to generate striated muscle fibers in vitro have been isolated from diverse adult tissues, although their physiological role being currently unclear. Since murine dermis-derived precursor cell cultures generate striated muscle when transplanted in vivo, we pursued to identify and characterize the myogenic cell population present in dermis-derived sphere cultures. Lineage tracing experiments for myogenic, perivascular and dermal precursor cell lineages showed a major contribution of Myf5 and Pax7-positive cell progeny to the dermal myogenic precursor cell subset. Tracing, in situ localization and ultrastructural analyses unequivocally demonstrated that Panniculus carnosus muscle-derived satellite stem cells expand in the dermal sphere culture conditions and originate dermis-derived myofibers in vitro. These results highlight the importance of unraveling distinct lineages in sphere cultures to avoid wrong assumptions when determining the developmental potential of adult stem cells. strain: Crl:CD1(ICR), B6.129S4-Myf5tm3(cre)Sor /J, B6,FVB-Tg(Cspg4-cre)1Akik/J, B195AP-Cre
Project description:Muscle stem cells, also known as satellite cells, represent the main myogenic population accounting for skeletal muscle homeostasis and regeneration. Here, using the Assay for Transposase Accessible Chromatin followed by sequencing (ATAC-seq), we investigated the epigenetic landscape of activated human and murine satellite cells. Our analysis identified a compendium of putative regulatory elements defining activated satellite cells and myoblasts, respectively.
Project description:Satellite cells (SC) are muscle stem cells which can regenerate adult muscles upon injury. Most SC originate from PAX7+ myogenic precursors set aside during development. Although myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced pluripotent stem cell (iPSC) reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPSC-derived PAX7+ myogenic precursors. We show that the PAX7+ cells generated in culture can produce myofibers and self-renew in vitro and in vivo. Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPSC, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.
Project description:The acquisition of a proliferating cell status from a quiescent state as well as the shift between proliferation and differentiation are key developmental steps in skeletal-muscle stem cells (satellite cells) to provide proper muscle regeneration. However, how satellite-cell proliferation is regulated, though, is not fully understood. Here, we report that the c-isoform of the transcription factor Pitx2 increases cell proliferation in myoblasts by down-regulating the miRNAs miR-15b, miR-23b, miR-106b, and miR-503. This Pitx2c-miRNA pathway also regulates cell proliferation in early-activated satellite cells, enhancing the Myf5+ satellite cells and thereby promoting their commitment to a myogenic cell fate. This study reveals unknown functions of several miRNAs in myoblast and satellite-cell behaviour and thus may have future applications in regenerative medicine. mirVana microarrays (Ambion) were used to profile microRNA signature at different Pitx2 overexpression conditions, namely two different doses (4 and 8 µg CMV-Pitx2c plasmid, respectively) after 24 hours of transfection in SOL8 skeletal myogenic cells. 20 µg of total RNA was used to hybridize two distinct microRNA microarrays on each condition analyzed.
Project description:To discover the molecules and signal pathways that are associated with the anti-aging effects of Fabp7 deficiency, transcriptome analyses were conducted using DNA microarray. The ABR thresholds of Fabp7 (+/+) and Fabp7 (-/-) mice were not significantly different at 7 months of age, but it was speculated that important gene expression changes might arise at approximately this stage. Therefore, 7-month-old Fabp7 (+/+) and Fabp7 (-/-) mice were used for transcriptome analyses.
Project description:The satellite cell is considered the major tissue-resident stem cell underlying muscle regeneration, however, multiple non-satellite cell myogenic progenitors have been identified. PW1/Peg3 is expressed in satellite cells as well as a subset of interstitial cells with myogenic potential termed PICs (PW1+ Interstitial Cells). PICs differ from satellite cells by their anatomical location (satellite cells are sublaminal and PICs are interstitial), they do not express any myogenic marker and arise from a Pax3-independent lineage. Upon isolation from juvenile muscle (1 to 3 weeks old), PICs are capable to form both skeletal and smooth muscle suggesting they constitute a more plastic population compared to satellite cells. We used microarrays to gain insight into the relantionship between PICs and satellite cells. PICs and satellite cells were isolated from 1-week old mouse muscle and subsequent RNA extraction was performed.
Project description:The objective of this study was to investigate the role of serine/arginine-rich splicing factor 1 (SRSF1) in the myogenic differentiation of bovine skeletal muscle satellite cells and its regulatory relationship with fibroblast growth factor 16 (FGF16). Bovine skeletal muscle satellite cells were subjected to SRSF1 knockdown and overexpression to evaluate their proliferation and differentiation capacities. RNA sequencing and subsequent validation experiments were performed to identify potential downstream targets of SRSF1, while functional assays were conducted to determine the role of FGF16 in myogenesis. RNA immunoprecipitation (RIP) and actinomycin D treatment were further used to investigate RNA-level interactions and transcript stability, and changes in PI3K/AKT signaling activity were assessed by western blotting. The results showed that SRSF1 expression dynamically increased during myogenic differentiation and significantly promoted myotube formation without markedly affecting cell proliferation. Transcriptomic analysis and validation identified FGF16 as a potential downstream target of SRSF1, and functional analyses demonstrated that FGF16 promoted myogenic differentiation. RIP assays suggested an association between SRSF1 mRNA and FGF16-associated complexes, while modulation of SRSF1 expression altered FGF16 expression and the phosphorylation status of PI3K and AKT. Collectively, these findings suggest that SRSF1 may promote the myogenic differentiation of bovine skeletal muscle satellite cells through post-transcriptional regulation associated with FGF16 and modulation of PI3K/AKT signaling activity, providing new insights into RNA-binding protein-mediated regulatory mechanisms involved in bovine skeletal muscle development.