Johnny Kim

Myf5-Positive Satellite Cells Contribute to Pax7-Dependent Long-Term Maintenance of Adult Muscle Stem Cells

Skeletal muscle contains Pax7-expressing muscle stem or satellite cells, enabling muscle regeneration throughout most of adult life. Here, we demonstrate that induced inactivation of Pax7 in Pax7-expressing cells of adult mice leads to loss of muscle stem cells and reduced heterochromatin condensation in rare surviving satellite cells. Inactivation of Pax7 in Myf5-expressing cells revealed that the majority of adult muscle stem cells originate from myogenic lineages, which express the myogenic regulators Myf5 or MyoD. Likewise, the majority of muscle stem cells are replenished from Myf5-expressing myogenic cells during adult life, and inactivation of Pax7 in Myf5-expressing cells after muscle damage leads to a complete arrest of muscle regeneration. Finally, we demonstrate that a relatively small number of muscle stem cells are sufficient for efficient repair of skeletal muscles. We conclude that Pax7 acts at different levels in a nonhierarchical regulatory network controlling muscle-satellite-cell-mediated muscle regeneration.

Prmt5 is a regulator of muscle stem cell expansion in adult mice

Skeletal muscle stem cells (MuSC), also called satellite cells, are indispensable for maintenance and regeneration of adult skeletal muscles. Yet, a comprehensive picture of the regulatory events controlling the fate of MuSC is missing. Here, we determine the proteome of MuSC to design a loss-of-function screen, and identify 120 genes important for MuSC function including the arginine methyltransferase Prmt5. MuSC-specific inactivation of Prmt5 in adult mice prevents expansion of MuSC, abolishes long-term MuSC maintenance and abrogates skeletal muscle regeneration. Interestingly, Prmt5 is dispensable for proliferation and differentiation of Pax7+ myogenic progenitor cells during mouse embryonic development, indicating significant differences between embryonic and adult myogenesis. Mechanistic studies reveal that Prmt5 controls proliferation of adult MuSC by direct epigenetic silencing of the cell cycle inhibitor p21. We reason that Prmt5 generates a poised state that keeps MuSC in a standby mode, thus allowing rapid MuSC amplification under disease conditions.

Epigenetic Modifications of Stem Cells: A Paradigm for the Control of Cardiac Progenitor Cells

Stem cells of all types are characterized by the ability to self-renew and to differentiate. Multiple lines of evidence suggest that both maintenance of stemness and lineage commitment, including determination of the cardiomyogenic lineage, are tightly controlled by epigenetic mechanisms such as DNA methylation, histone modifications, and ATP-dependent chromatin remodeling. Epigenetic mechanisms are intrinsically reversible, interdependent, and highly dynamic in regulation of chromatin structure and specific gene transcription programs, thereby contributing to stem cell homeostasis. Here, we review the current understanding of epigenetic mechanisms involved in regulation of stem cell self-renewal and differentiation and in the control of cardiac progenitor cell commitment during heart development. Further progress in this area will help to decipher the epigenetic landscape in stem and progenitor cells and facilitate manipulation of stem cells for regenerative applications.

Neurofibromin (Nf1) is required for skeletal muscle development

Neurofibromatosis type 1 (NF1) is a multi-system disease caused by mutations in the NF1 gene encoding a Ras-GAP protein, neurofibromin, which negatively regulates Ras signaling. Besides neuroectodermal malformations and tumors, the skeletal system is often affected (e.g. scoliosis and long bone dysplasia) demonstrating the importance of neurofibromin for development and maintenance of the musculoskeletal system. Here, we focus on the role of neurofibromin in skeletal muscle development. Nf1 gene inactivation in the early limb bud mesenchyme using Prx1-cre (Nf1(Prx1)) resulted in muscle dystrophy characterized by fibrosis, reduced number of muscle fibers and reduced muscle force. This was caused by an early defect in myogenesis affecting the terminal differentiation of myoblasts between E12.5 and E14.5. In parallel, the muscle connective tissue cells exhibited increased proliferation at E14.5 and an increase in the amount of connective tissue as early as E16.5. These changes were accompanied by excessive mitogen-activated protein kinase pathway activation. Satellite cells isolated from Nf1(Prx1) mice showed normal self-renewal, but their differentiation was impaired as indicated by diminished myotube formation. Our results demonstrate a requirement of neurofibromin for muscle formation and maintenance. This previously unrecognized function of neurofibromin may contribute to the musculoskeletal problems in NF1 patients.

Overexpression of Twinkle-helicase protects cardiomyocytes from genotoxic stress caused by reactive oxygen species

Significance In the present work, we show that overexpression of TWINKLE helicase reduces the amount of ROS-induced mtDNA mutations and ameliorates cardiomyopathy in Sod2+/− mice. We demonstrate that increased ROS in mitochondria result in a rise of base transversions and mtDNA rearrangements. Increased TWINKLE availability improves mtDNA integrity and protects cardiomyocytes by inhibiting apoptosis via p21. Our findings offer unique approaches to limit the loss of cardiomyocytes due to oxidative stress, a common problem in various disease conditions and during normal aging. Mitochondrial DNA (mtDNA) in adult human heart is characterized by complex molecular forms held together by junctional molecules of unknown biological significance. These junctions are not present in mouse hearts and emerge in humans during postnatal development, concomitant with increased demand for oxidative metabolism. To analyze the role of mtDNA organization during oxidative stress in cardiomyocytes, we used a mouse model, which recapitulates the complex mtDNA organization of human hearts by overexpression of the mitochondrial helicase, TWINKLE. Overexpression of TWINKLE rescued the oxidative damage induced replication stalling of mtDNA, reduced mtDNA point mutation load, and modified mtDNA rearrangements in heterozygous mitochondrial superoxide dismutase knockout hearts, as well as ameliorated cardiomyopathy in mice superoxide dismutase knockout in a p21-dependent manner. We conclude that mtDNA integrity influences cell survival and reason that tissue specific modes of mtDNA maintenance represent an adaptation to oxidative stress.

The Emerging Role of Epigenetic Modifiers Linking Cellular Metabolism and Gene Activity in Cardiac Progenitor Cells

During mammalian heart development, cardiac gene expression is controlled by a complex network consisting of signaling pathways, cardiac transcription factors, and epigenetic modifiers. Emerging evidence suggests that epigenetic modifying enzymes sense and respond to metabolic cues, thereby translating environmental stimuli to cardiac gene expression patterns. Here, we review the impact of metabolic cues on epigenetic changes and survey how epigenetic changes, including DNA modifications, histone modifications, and ATP-dependent chromatin remodeling, affect recruitment of progenitor cells into the cardiac lineage. We reason that a better understanding of epigenetic control mechanisms regulating cardiac gene expression will improve reprogramming strategies to generate cardiovascular cells for therapeutic applications.

Targeting the Cellular Origin of Organ Fibrosis

Organ fibrosis after injury, which can significantly compromise tissue function, is in part due to proliferation of fibroblasts and myofibroblasts of unclear cellular origin. Now in Cell Stem Cell, Kramann et al. (2015) show that perivascular Gli1+ mesenchymal-stem-cell-like cells generate a large fraction of myofibroblasts in disease conditions.

Systematic Identification of Genes Regulating Muscle Stem Cell Self-Renewal and Differentiation

The hallmark of stem cells is their capability to either self-renew or to differentiate into a different cell type. Adult skeletal muscle contains a resident muscle stem cell population (MuSCs) known as satellite cells, which enables regeneration of damaged muscle tissue throughout most of adult life. During skeletal muscle regeneration, few MuSCs self-renew to maintain the muscle stem cell pool while others expand rapidly and subsequently undergo myogenic differentiation to form new myofibers. However, like for other stem cell types, the molecular networks that govern self-renewal and/or differentiation of MuSCs remain largely elusive. We recently reported a method to isolate sufficient amounts of purified MuSCs from skeletal muscle which enables us to study their cell autonomous properties. Here, we describe a lentiviral, image-based loss-of function screening pipeline on primary MuSCs that enables systematic identification of genes that regulate muscle stem cell function.

Skeletal muscle stem cells for muscle regeneration.

Adult skeletal muscle possesses remarkable regenerative capacity. Muscle regeneration is mediated by a rare population of muscle stem cells that reside between the basal lamina and sarcolemma of muscle fibers. Due to their anatomical location, muscle stem cells have been coined satellite cells. Here, we describe a method that we routinely use to isolate large and pure populations of satellite cells from skeletal muscles enabling studies on autonomous properties of satellite cells to unravel the role of muscle stem cells in tissue regeneration.

RNA-Seq analysis of isolated satellite cells in Prmt5 deficient mice

Satellite cells (SCs) represent a distinct population of stem cells, essential for maintenance, growth and regeneration of adult skeletal muscle. SCs are mononuclear and are located between the basal lamina and the plasma membrane of myofibers. They are typically characterized by presence of the transcription factor paired-box 7 (PAX7) that is widely used as a satellite cell marker. Under normal physiological conditions SCs are quiescent but are activated by insults such as injury, disease or exercise. Once activated, satellite cells proliferate and subsequently differentiate into myoblasts to finally fuse to form new myofibers or with preexisting myofibers to repair or rebuild the skeletal muscle. A minority of SCs retains stem cell characteristics and self-renews to assure future bouts of regeneration throughout most of adult life. While a comprehensive picture of the regulatory events controlling SC fate has not yet been achieved, several factors were recently identified playing important roles in functional processes. One example is the arginine methyltransferase Prmt5 that is known to have multiple roles in germ cells and is involved in the maintenance of ES cell pluripotency. We have previously shown that Prmt5 is required for muscle stem cell proliferation and regenerative myogenesis due to direct epigenetic regulation of the cell cycle inhibitor p21. Here we provide a dataset that investigates the loss of Prmt5 in isolated Pax7+ primary SCs using the Pax7CreERT2/Prmt5loxP/loxP knockout mouse model. RNA-Seq raw and analyzed data have been deposited in GEO under accession code GSE66822.