Project description:Mouse muscle stem cells, defined as Pax7+ satellite cells, can initiate rhabdomyosarcoma when transformed by oncogenic Kras and concomitant loss of p53. Mouse Pax7+ satellite cells were transformed in vitro and in vivo utilizing the Cre-ER/loxp system. We wanted to address two major questions: do the in vitro and in vivo tumors cluster together compared to another mouse to another mouse derived soft-tissue sarcoma AND which human soft-tissue sarcoma do the in vivo derived tumors resemble transcriptionally? Therefore, tumors from cells transformed in vitro and tumors from mice that restrict the oncogenic lesions to Pax7+ satellite cells in vivo were compared to answer these two questions.
Project description:Mouse muscle stem cells, defined as Pax7+ satellite cells, can initiate rhabdomyosarcoma when transformed by oncogenic Kras and concomitant loss of p53. Mouse Pax7+ satellite cells were transformed in vitro and in vivo utilizing the Cre-ER/loxp system.
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:Pax7 is a paired box transcription factor that is central to skeletal muscle satellite cell function Microarrays were used to examine gene expression after primary murine satellite cell-derived myoblasts were retrovirally infected with constructs encoding Pax7, PAX7-FOXO1A, dominant negative Pax7-ERD and retroviral control.
Project description:Quiescent muscle stem cells, commonly known as satellite cells, are crucial for muscle repair and can convert into committed myoblasts capable of proliferation and differentiation upon in vitro culture. However, following prolonged propagation, myoblasts frequently lose myogenic differentiation capacity, limiting their utility in research and clinical applications. Here, we demonstrate that exposing committed mouse myoblasts to a small-molecule cocktail elicits their conversion into expandable and heterogeneous myogenic progenitor cells (MPCs), comprised of muscle stem, progenitor and differentiated cells. Utilizing a new dual-fluorescent reporter for Pax7 and MyoD, we demonstrate that the small molecules de-differentiate Pax7+/MyoD+ myoblasts into Pax7+/MyoD- satellite-like cells within days. This conversion is characterized by upregulation of signaling pathways associated with satellite cells including Notch, Calcitonin and EGFR. Accordingly, genetic ablation of Notch1-expressing cells abrogated MPC cultures but not committed myoblasts. Furthermore, a comparison with in vivo-derived freshly isolated and activated satellite cells through single-cell transcriptomics revealed that the stem cell subset in MPCs shares common features with both cell types, particularly with a sub-population of activated satellite cells. Collectively, our study presents a method to de-differentiate myoblasts into MPCs harboring satellite cell attributes in vitro, offering a new avenue for studying myogenesis and advancing muscle disease therapeutics.
Project description:Quiescent muscle stem cells, commonly known as satellite cells, are crucial for muscle repair and can convert into committed myoblasts capable of proliferation and differentiation upon in vitro culture. However, following prolonged propagation, myoblasts frequently lose myogenic differentiation capacity, limiting their utility in research and clinical applications. Here, we demonstrate that exposing committed mouse myoblasts to a small-molecule cocktail elicits their conversion into expandable and heterogeneous myogenic progenitor cells (MPCs), comprised of muscle stem, progenitor and differentiated cells. Utilizing a new dual-fluorescent reporter for Pax7 and MyoD, we demonstrate that the small molecules de-differentiate Pax7+/MyoD+ myoblasts into Pax7+/MyoD- satellite-like cells within days. This conversion is characterized by upregulation of signaling pathways associated with satellite cells including Notch, Calcitonin and EGFR. Accordingly, genetic ablation of Notch1-expressing cells abrogated MPC cultures but not committed myoblasts. Furthermore, a comparison with in vivo-derived freshly isolated and activated satellite cells through single-cell transcriptomics revealed that the stem cell subset in MPCs shares common features with both cell types, particularly with a sub-population of activated satellite cells. Collectively, our study presents a method to de-differentiate myoblasts into MPCs harboring satellite cell attributes in vitro, offering a new avenue for studying myogenesis and advancing muscle disease therapeutics.
Project description:Optimal cell-based therapies for the treatment of muscle degenerative disorders should not only regenerate fibers, but provide a quiescent satellite cell pool ensuring long-term maintenance and regeneration. Conditional expression of Pax3/Pax7 in differentiating pluripotent stem cells (PSC) allows the generation of myogenic progenitors endowed with satellite cell-like abilities. To identify the molecular determinants underlying their regenerative potential, we performed transcriptome analyses of these cells along with primary myogenic cells from several developmental stages. Here we show that in vitro generated PSC-derived myogenic progenitors possess a molecular signature similar to embryonic/fetal myoblasts. However, compared to fetal myoblasts, following transplantation they show superior myofiber engraftment and ability to seed the satellite cell niche, respond to multiple re-injuries and contribute to long-term regeneration. Upon engraftment, the transcriptome of Pax3/Pax7-induced PSC-derived myogenic progenitors changes dramatically, acquiring similarity to that of satellite cells, particularly in genes involved in extracellular matrix remodeling. Single cell profiling reveals that these changes are induced, not selected, by the in vivo environment. These findings demonstrate that Pax3/Pax7-induced PSC-derived myogenic progenitors possess proliferative and migratory abilities characteristic of earlier developmental stages, and an intrinsic ability to respond to environmental cues upon skeletal muscle regeneration.
Project description:Optimal cell-based therapies for the treatment of muscle degenerative disorders should not only regenerate fibers, but provide a quiescent satellite cell pool ensuring long-term maintenance and regeneration. Conditional expression of Pax3/Pax7 in differentiating pluripotent stem cells (PSC) allows the generation of myogenic progenitors endowed with satellite cell-like abilities. To identify the molecular determinants underlying their regenerative potential, we performed transcriptome analyses of these cells along with primary myogenic cells from several developmental stages. Here we show that in vitro generated PSC-derived myogenic progenitors possess a molecular signature similar to embryonic/fetal myoblasts. However, compared to fetal myoblasts, following transplantation they show superior myofiber engraftment and ability to seed the satellite cell niche, respond to multiple re-injuries and contribute to long-term regeneration. Upon engraftment, the transcriptome of Pax3/Pax7-induced PSC-derived myogenic progenitors changes dramatically, acquiring similarity to that of satellite cells, particularly in genes involved in extracellular matrix remodeling. Single cell profiling reveals that these changes are induced, not selected, by the in vivo environment. These findings demonstrate that Pax3/Pax7-induced PSC-derived myogenic progenitors possess proliferative and migratory abilities characteristic of earlier developmental stages, and an intrinsic ability to respond to environmental cues upon skeletal muscle regeneration.
Project description:Optimal cell-based therapies for the treatment of muscle degenerative disorders should not only regenerate fibers, but provide a quiescent satellite cell pool ensuring long-term maintenance and regeneration. Conditional expression of Pax3/Pax7 in differentiating pluripotent stem cells (PSC) allows the generation of myogenic progenitors endowed with satellite cell-like abilities. To identify the molecular determinants underlying their regenerative potential, we performed transcriptome analyses of these cells along with primary myogenic cells from several developmental stages. Here we show that in vitro generated PSC-derived myogenic progenitors possess a molecular signature similar to embryonic/fetal myoblasts. However, compared to fetal myoblasts, following transplantation they show superior myofiber engraftment and ability to seed the satellite cell niche, respond to multiple re-injuries and contribute to long-term regeneration. Upon engraftment, the transcriptome of Pax3/Pax7-induced PSC-derived myogenic progenitors changes dramatically, acquiring similarity to that of satellite cells, particularly in genes involved in extracellular matrix remodeling. Single cell profiling reveals that these changes are induced, not selected, by the in vivo environment. These findings demonstrate that Pax3/Pax7-induced PSC-derived myogenic progenitors possess proliferative and migratory abilities characteristic of earlier developmental stages, and an intrinsic ability to respond to environmental cues upon skeletal muscle regeneration.