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Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro.

ABSTRACT: Satellite cells (SC) are muscle stem cells that 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.

SUBMITTER: Al Tanoury Z

PROVIDER: S-EPMC7328153 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro

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.

2020-06-15 | GSE149451 | GEO

Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro

Project description:Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro

| PRJNA628701 | ENA

Characterization of the Methylation Status of Pax7 and Myogenic Regulator Factors in Cell Myogenic Differentiation.

Project description:Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

| S-EPMC4932581 | biostudies-literature

Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification.

Project description:The paired-box transcription factor Pax7 has been claimed to specify the muscle stem cell lineage since inactivation of Pax7 led to a failure to detect muscle satellite cells. Here we show that muscles of juvenile Pax7(-/-) mice at P11 contain a reduced but substantial number of satellite cells. Neither juvenile nor adult Pax7(-/-) mice displayed a significant reduction in the number and size of myotubes, indicating that the remaining number of satellite cells sufficed to allow normal postnatal muscle growth. The number of satellite cells in Pax7 mutant mice declined strongly during postnatal development, although single satellite cells were readily identified in adult Pax7 mutant mice. Muscle regeneration was impaired in adult Pax7 mutant mice. Our results clearly indicate an essential function of Pax7 for renewal and maintenance of muscle stem cells and exclude an exclusive role of Pax7 in satellite cell specification.

| S-EPMC514519 | biostudies-literature

Barx2 and Pax7 have antagonistic functions in regulation of wnt signaling and satellite cell differentiation.

Project description:The canonical Wnt signaling pathway is critical for myogenesis and can induce muscle progenitors to switch from proliferation to differentiation; how Wnt signals integrate with muscle-specific regulatory factors in this process is poorly understood. We previously demonstrated that the Barx2 homeobox protein promotes differentiation in cooperation with the muscle regulatory factor (MRF) MyoD. Pax7, another important muscle homeobox factor, represses differentiation. We now identify Barx2, MyoD, and Pax7 as novel components of the Wnt effector complex, providing a new molecular pathway for regulation of muscle progenitor differentiation. Canonical Wnt signaling induces Barx2 expression in muscle progenitors and perturbation of Barx2 leads to misregulation of Wnt target genes. Barx2 activates two endogenous Wnt target promoters as well as the Wnt reporter gene TOPflash, the latter synergistically with MyoD. Moreover, Barx2 interacts with the core Wnt effectors ?-catenin and T cell-factor 4 (TCF4), is recruited to TCF/lymphoid enhancer factor sites, and promotes recruitment of ?-catenin. In contrast, Pax7 represses the Wnt reporter gene and antagonizes the activating effect of Barx2. Pax7 also binds ?-catenin suggesting that Barx2 and Pax7 may compete for interaction with the core Wnt effector complex. Overall, the data show for the first time that Barx2, Pax7, and MRFs can act as direct transcriptional effectors of Wnt signals in myoblasts and that Barx2 and Wnt signaling participate in a regulatory loop. We propose that antagonism between Barx2 and Pax7 in regulation of Wnt signaling may help mediate the switch from myoblast proliferation to differentiation.

| S-EPMC4134105 | biostudies-literature

Integrated functions of Pax3 and Pax7 in the regulation of proliferation, cell size and myogenic differentiation.

Project description:Pax3 and Pax7 are paired-box transcription factors with roles in developmental and adult regenerative myogenesis. Pax3 and Pax7 are expressed by postnatal satellite cells or their progeny but are down regulated during myogenic differentiation. We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size. Conversely, expression of dominant-negative constructs leads to slowing of cell division, a dramatic increase in cell size and altered morphology. Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation. However, expression of Pax3 or Pax7 dominant-negative constructs inhibits expression of Myf5, MyoD and myogenin, and prevents differentiation from proceeding. In fibroblasts, expression of Pax3 or Pax7, or dominant-negative inhibition of these factors, reproduce the effects on cell size, morphology and proliferation seen in myoblasts. Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed.

| S-EPMC2637421 | biostudies-literature

Smad3 signaling is required for satellite cell function and myogenic differentiation of myoblasts.

Project description:TGF-? and myostatin are the two most important regulators of muscle growth. Both growth factors have been shown to signal through a Smad3-dependent pathway. However to date, the role of Smad3 in muscle growth and differentiation is not investigated. Here, we demonstrate that Smad3-null mice have decreased muscle mass and pronounced skeletal muscle atrophy. Consistent with this, we also find increased protein ubiquitination and elevated levels of the ubiquitin E3 ligase MuRF1 in muscle tissue isolated from Smad3-null mice. Loss of Smad3 also led to defective satellite cell (SC) functionality. Smad3-null SCs showed reduced propensity for self-renewal, which may lead to a progressive loss of SC number. Indeed, decreased SC number was observed in skeletal muscle from Smad3-null mice showing signs of severe muscle wasting. Further in vitro analysis of primary myoblast cultures identified that Smad3-null myoblasts exhibit impaired proliferation, differentiation and fusion, resulting in the formation of atrophied myotubes. A search for the molecular mechanism revealed that loss of Smad3 results in increased myostatin expression in Smad3-null muscle and myoblasts. Given that myostatin is a negative regulator, we hypothesize that increased myostatin levels are responsible for the atrophic phenotype in Smad3-null mice. Consistent with this theory, inactivation of myostatin in Smad3-null mice rescues the muscle atrophy phenotype.

| S-EPMC3364732 | biostudies-literature

Myogenic Differentiation of Mouse Embryonic Stem Cells That Lack a Functional Pax7 Gene.

Project description:The transcription factor Pax7 plays a key role during embryonic myogenesis and sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Overexpression of Pax7 has been shown to promote the myogenic differentiation of pluripotent stem cells. However, the effects of the absence of functional Pax7 in differentiating embryonic stem cells (ESCs) have not yet been directly tested. Herein, we studied mouse stem cells that lacked a functional Pax7 gene and characterized the differentiation of these stem cells under conditions that promoted the derivation of myoblasts in vitro. We analyzed the expression of myogenic factors, such as myogenic regulatory factors and muscle-specific microRNAs, in wild-type and mutant cells. Finally, we compared the transcriptome of both types of cells and did not find substantial differences in the expression of genes related to the regulation of myogenesis. As a result, we showed that the absence of functional Pax7 does not prevent the in vitro myogenic differentiation of ESCs.

| S-EPMC4761802 | biostudies-literature

Impaired regeneration in LGMD2A supported by increased PAX7-positive satellite cell content and muscle-specific microrna dysregulation.

Project description:Recent in vitro studies suggest that CAPN3 deficiency leads initially to accelerated myofiber formation followed by depletion of satellite cells (SC). In normal muscle, up-regulation of miR-1 and miR-206 facilitates transition from proliferating SCs to differentiating myogenic progenitors.We examined the histopathological stages, Pax7 SC content, and muscle-specific microRNA expression in biopsy specimens from well-characterized LGMD 2A patients to gain insight into disease pathogenesis.Three distinct stages of pathological changes were identified that represented the continuum of the dystrophic process from prominent inflammation with necrosis and regeneration to prominent fibrosis, which correlated with age and disease duration. Pax7-positive SCs were highest in the fibrotic group and correlated with down-regulation of miR-1, miR-133a, and miR-206.These observations, and other published reports, are consistent with microRNA dysregulation leading to inability of Pax7-positive SCs to transit from proliferation to differentiation. This results in impaired regeneration and fibrosis.

| S-EPMC3634894 | biostudies-literature

Dermal myogenic precursors derive from muscle satellite stem cells

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

2017-02-21 | GSE67693 | GEO

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