tumorsphere-derivation-treatment-from-primary-tumor-cells-isolated

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

Sanford Burnham Prebys Medical Discovery Institute

Muscle stem cells (MuSCs) exhibit distinct behavior during successive phases of developmental myogenesis. However, how their transition to adulthood is regulated is poorly understood. Here, we show that fetal MuSCs resist progenitor specification and exhibit altered division dynamics, intrinsic features that are progressively lost postnatally. After transplantation, fetal MuSCs expand more efficiently and contribute to muscle repair. Conversely, niche colonization efficiency increases in adulthood, indicating a balance between muscle growth and stem cell pool repopulation. Gene expression profiling identified several extracellular matrix (ECM) molecules preferentially expressed in fetal MuSCs, including tenascin-C, fibronectin, and collagen VI. Loss-of-function experiments confirmed their essential and stage-specific role in regulating MuSC function. Finally, fetal-derived paracrine factors were able to enhance adult MuSC regenerative potential. Together, these findings demonstrate that MuSCs change the way in which they remodel their microenvironment to direct stem cell behavior and support the unique demands of muscle development or repair. 

Autonomous Extracellular Matrix Remodeling Controls a Progressive Adaptation in Muscle Stem Cell Regenerative Capacity during Development.

The cellular turnover required for skeletal muscle maintenance and repair is mediated by resident stem cells, also termed satellite cells. Satellite cells normally reside in a quiescent state, intermittently entering the cell cycle to fuse with neighboring myofibers and replenish the stem cell pool. However, the mechanisms by which satellite cells maintain the precise balance between self-renewal and differentiation necessary for long-term homeostasis remain unclear. Recent work has supported a previously unappreciated heterogeneity in the satellite cell compartment that may underlie the observed variability in cell fate and function. In this review, we examine the work supporting this notion as well as the potential governing principles, developmental origins, and principal determinants of satellite cell heterogeneity.

Satellite Cell Heterogeneity in Skeletal Muscle Homeostasis.

Duchenne muscular dystrophy (DMD) is caused by loss of the structural protein, dystrophin, resulting in muscle fragility. Muscle stem cell (MuSC) transplantation is a potential therapy for DMD. It is unknown whether donor-derived muscle fibers are structurally innervated.

Innervation of dystrophic muscle after muscle stem cell therapy.

The review summarizes our current knowledge of the role of signal transducer and activator of transcription 3 (STAT3) signaling in skeletal muscle regeneration and the maintenance of muscle mass.

Signal transducer and activator of transcription 3 signaling as a potential target to treat muscle wasting diseases.

The role of muscle stem cell-niche interactions during aging.

Models of skeletal muscle injury in animal models are invaluable tools to assess muscle stem cell (MuSC)-mediated tissue repair. The optimization and comprehensive evaluation of these approaches have greatly improved our ability to assess MuSC regenerative potential. Here we describe the procedures for skeletal muscle injury with notexin and BaCl2 and assessment of the dynamics of tissue regeneration.

Inducing and Evaluating Skeletal Muscle Injury by Notexin and Barium Chloride.

Skeletal muscle stem cell (MuSC) isolation and transplantation are invaluable tools to assess their capacity for self-renewal and tissue repair. Significant technical advances in recent years have led to the optimization of these approaches, improving our ability to assess MuSC regenerative potential. Here, we describe the procedures for Fluorescent Activated Cell Sorting (FACS)-based isolation of MuSC, their intramuscular transplantation, and analysis of their engraftment into host tissues.

Engraftment of FACS Isolated Muscle Stem Cells into Injured Skeletal Muscle.

Injection of skeletal muscle progenitors has the potential to be a minimally invasive treatment for a number of diseases that negatively affect vasculature and skeletal muscle, including peripheral artery disease. However, success with this approach has been limited because of poor transplant cell survival. This is primarily attributed to cell death due to extensional flow through the needle, the harsh ischemic environment of the host tissue, a deleterious immune cell response, and a lack of biophysical cues supporting exogenous cell viability. We show that engineering a muscle-specific microenvironment, using a nanofibrous decellularized skeletal muscle extracellular matrix hydrogel and skeletal muscle fibroblasts, improves myoblast viability and maturation in vitro. In vivo, this translates to improved cell survival and engraftment and increased perfusion as a result of increased vascularization. Our results indicate that a combinatorial delivery system, which more fully recapitulates the tissue microenvironment, can improve cell delivery to skeletal muscle.

Engineering an Injectable Muscle-Specific Microenvironment for Improved Cell Delivery Using a Nanofibrous Extracellular Matrix Hydrogel.

The molecular determinants of muscle progenitor impairment to regenerate aged muscles are currently unclear. We show that, in a mouse model of replicative senescence, decline in muscle satellite cell-mediated regeneration coincides with activation of DNA damage response (DDR) and impaired ability to differentiate into myotubes. Inhibition of DDR restored satellite cell differentiation ability. Moreover, in replicative human senescent fibroblasts, DDR precluded MYOD-mediated activation of the myogenic program. A DDR-resistant MYOD mutant could overcome this barrier by resuming cell cycle progression. Likewise, DDR inhibition could also restore MYOD's ability to activate the myogenic program in human senescent fibroblasts. Of note, we found that cell cycle progression is necessary for the DDR-resistant MYOD mutant to reverse senescence-mediated inhibition of the myogenic program. These data provide the first evidence of DDR-mediated functional antagonism between senescence and MYOD-activated gene expression and indicate a previously unrecognized requirement of cell cycle progression for the activation of the myogenic program.

DNA damage signaling mediates the functional antagonism between replicative senescence and terminal muscle differentiation.

Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix-loop-helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation-Tcf3 and Foxa2-and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1-4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.

Id genes are essential for early heart formation.

Muscle stem cells (MuSCs) contribute to muscle regeneration following injury. In many muscle disorders, the repeated cycles of damage and repair lead to stem cell dysfunction. While telomere attrition may contribute to aberrant stem cell functions, methods to accurately measure telomere length in stem cells from skeletal muscles have not been demonstrated. Here, we have optimized and validated such a method, named MuQ-FISH, for analyzing telomere length in MuSCs from either mice or humans. Our analysis showed no differences in telomere length between young and aged MuSCs from uninjured wild-type mice, but MuSCs isolated from young dystrophic mice exhibited significantly shortened telomeres. In corroboration, we demonstrated that telomere attrition is present in human dystrophic MuSCs, which underscores its importance in diseased regenerative failure. The robust technique described herein provides analysis at a single-cell resolution and may be utilized for other cell types, especially rare populations of cells.

Single Stem Cell Imaging and Analysis Reveals Telomere Length Differences in Diseased Human and Mouse Skeletal Muscles.

The clonal complexity of adult stem cell pools is progressively lost during homeostatic turnover in several tissues, suggesting a decrease in the number of stem cells with distinct clonal origins. The functional impact of reduced complexity on stem cell pools, and how different tissue microenvironments may contribute to such a reduction, are poorly understood. Here, we performed clonal multicolor lineage tracing of skeletal muscle stem cells (MuSCs) to address these questions. We found that MuSC clonal complexity is maintained during aging despite heterogenous reductions in proliferative capacity, allowing aged muscle to mount a clonally diverse, albeit diminished, response to injury. In contrast, repeated bouts of tissue repair cause a progressive reduction in MuSC clonal complexity indicative of neutral drift. Consistently, biostatistical modeling suggests that MuSCs undergo symmetric expansions with stochastic fate acquisition during tissue repair. These findings establish distinct principles that underlie stem cell dynamics during homeostatic aging and muscle regeneration.

Muscle Stem Cells Exhibit Distinct Clonal Dynamics in Response to Tissue Repair and Homeostatic Aging.

Changes in muscle stem cell (MuSC) function during aging have been assessed using various in vivo and ex vivo systems. However, changes in clonal complexity within the aged MuSC pool are relatively understudied. Although the dissection of stem cell heterogeneity has greatly benefited from several technological advancements, including single cell sequencing, these methods preclude longitudinal measures of individual stem cell behavior. Instead, multicolor labeling systems enable lineage tracing with single cell resolution. Here, we describe a method of inducibly labeling MuSCs with the Brainbow-2.1 multicolor lineage tracing reporter in vivo to track individual MuSC fate and assess clonal complexity in the overall MuSC pool throughout the mouse lifespan.

Assessing Muscle Stem Cell Clonal Complexity During Aging.

Fibro-adipogenic progenitors (FAPs) are typically activated in response to muscle injury, and establish functional interactions with inflammatory and muscle stem cells (MuSCs) to promote muscle repair. We found that denervation causes progressive accumulation of FAPs, without concomitant infiltration of macrophages and MuSC-mediated regeneration. Denervation-activated FAPs exhibited persistent STAT3 activation and secreted elevated levels of IL-6, which promoted muscle atrophy and fibrosis. FAPs with aberrant activation of STAT3-IL-6 signalling were also found in mouse models of spinal cord injury, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS) and in muscles of ALS patients. Inactivation of STAT3-IL-6 signalling in FAPs effectively countered muscle atrophy and fibrosis in mouse models of acute denervation and ALS (SOD mice). Activation of pathogenic FAPs following loss of integrity of neuromuscular junctions further illustrates the functional versatility of FAPs in response to homeostatic perturbations and suggests their potential contribution to the pathogenesis of neuromuscular diseases.

Denervation-activated STAT3-IL-6 signalling in fibro-adipogenic progenitors promotes myofibres atrophy and fibrosis.

Most human cancers originate from high-turnover tissues, while low-proliferating tissues, like skeletal muscle, exhibit a lower incidence of tumor development. In Duchenne muscular dystrophy (DMD), which induces increased skeletal muscle regeneration, tumor incidence is increased. Rhabdomyosarcomas (RMSs), a rare and aggressive type of soft tissue sarcoma, can develop in this context, but the impact of DMD severity on RMS development and its cell of origin are poorly understood. Here, we show that RMS latency is affected by DMD severity and that muscle stem cells (MuSCs) can give rise to RMS in dystrophic mice. We report that even before tumor formation, MuSCs exhibit increased self-renewal and an expression signature associated with RMSs. These cells can form tumorspheres in vitro and give rise to RMSs in vivo. Finally, we show that the inflammatory genes Ccl11 and Rgs5 are involved in RMS growth. Together, our results show that DMD severity drives MuSC-mediated RMS development.

Muscle Stem Cells Give Rise to Rhabdomyosarcomas in a Severe Mouse Model of Duchenne Muscular Dystrophy.

Metabolic reprogramming is an active regulator of stem cell fate choices, and successful stem cell differentiation in different compartments requires the induction of oxidative phosphorylation. However, the mechanisms that promote mitochondrial respiration during stem cell differentiation are poorly understood. Here we demonstrate that Stat3 promotes muscle stem cell myogenic lineage progression by stimulating mitochondrial respiration in mice. We identify Fam3a, a cytokine-like protein, as a major Stat3 downstream effector in muscle stem cells. We demonstrate that Fam3a is required for muscle stem cell commitment and skeletal muscle development. We show that myogenic cells secrete Fam3a, and exposure of Stat3-ablated muscle stem cells to recombinant Fam3a in vitro and in vivo rescues their defects in mitochondrial respiration and myogenic commitment. Together, these findings indicate that Fam3a is a Stat3-regulated secreted factor that promotes muscle stem cell oxidative metabolism and differentiation, and suggests that Fam3a is a potential tool to modulate cell fate choices.

Article	Year
Autonomous Extracellular Matrix Remodeling Controls a Progressive Adaptation in Muscle Stem Cell Regenerative Capacity during Development. Cell reports\| PubMed ID: 26904948	2016
Satellite Cell Heterogeneity in Skeletal Muscle Homeostasis. Trends in cell biology\| PubMed ID: 26948993	2016
Innervation of dystrophic muscle after muscle stem cell therapy. Muscle & nerve\| PubMed ID: 26998682	2016
Signal transducer and activator of transcription 3 signaling as a potential target to treat muscle wasting diseases. Current opinion in clinical nutrition and metabolic care\| PubMed ID: 27023048	2016
The role of muscle stem cell-niche interactions during aging. Nature medicine\| PubMed ID: 27490436	2016
Inducing and Evaluating Skeletal Muscle Injury by Notexin and Barium Chloride. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 27492165	2016
Engraftment of FACS Isolated Muscle Stem Cells into Injured Skeletal Muscle. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28247352	2017
Engineering an Injectable Muscle-Specific Microenvironment for Improved Cell Delivery Using a Nanofibrous Extracellular Matrix Hydrogel. ACS nano\| PubMed ID: 28323411	2017
DNA damage signaling mediates the functional antagonism between replicative senescence and terminal muscle differentiation. Genes & development\| PubMed ID: 28446595	2017
Id genes are essential for early heart formation. Genes & development\| PubMed ID: 28794185	2017
Single Stem Cell Imaging and Analysis Reveals Telomere Length Differences in Diseased Human and Mouse Skeletal Muscles. Stem cell reports\| PubMed ID: 28890163	2017
Muscle Stem Cells Exhibit Distinct Clonal Dynamics in Response to Tissue Repair and Homeostatic Aging. Cell stem cell\| PubMed ID: 29249462	2018
Assessing Muscle Stem Cell Clonal Complexity During Aging. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29845579	2019
Denervation-activated STAT3-IL-6 signalling in fibro-adipogenic progenitors promotes myofibres atrophy and fibrosis. Nature cell biology\| PubMed ID: 30050118	2018
Muscle Stem Cells Give Rise to Rhabdomyosarcomas in a Severe Mouse Model of Duchenne Muscular Dystrophy. Cell reports\| PubMed ID: 30650360	2019
The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration. Nature communications\| PubMed ID: 30996264	2019

Alessandra Sacco

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