Abstract This unit describes the use of PCR to characterize and quantify rearranged transcripts from specific T cell receptor variable gene families in human tissue and peripheral blood lymphocytes. The strategy outlined in this unit has been extensively used on different sources of human tissue including brain, spinal cord, and skeletal muscle. A protocol is provided to clone and sequence PCR‐amplified cDNA transcripts to study the junctional diversity of the expressed genes. A support protocol describes a method for reverse transcribing total RNA to make the cDNA required by the other protocols.
Severe childhood autosomal recessive muscular dystrophy (SCARMD) is a progressive muscle-wasting disorder common in North Africa that segregates with microsatellite markers at chromosome 13q12. Here, it is shown that a mutation in the gene encoding the 35-kilodalton dystrophin-associated glycoprotein, γ-sarcoglycan, is likely to be the primary genetic defect in this disorder. The human γ-sarcoglycan gene was mapped to chromosome 13q12, and deletions that alter its reading frame were identified in three families and one of four sporadic cases of SCARMD. These mutations not only affect γ-sarcoglycan but also disrupt the integrity of the entire sarcoglycan complex.
Satellite cells (SC) are muscle stem cells which can regenerate adult muscles upon injury. Most SC originate from PAX7-positive myogenic precursors set aside during development. While 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 (iPS) cell 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 iPS-derived PAX7-positive myogenic precursors. We show that the PAX7-positive 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 iPS cells, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.
Abstract Stem cell transplantation is being tested as a potential therapy for a number of diseases. Stem cells isolated directly from tissue specimens or generated via reprogramming of differentiated cells require rigorous testing for both safety and efficacy in preclinical models. The availability of mice with immune-deficient background that carry additional mutations in specific genes facilitates testing the efficacy of cell transplantation in disease models. The muscular dystrophies are a heterogeneous group of disorders, of which Duchenne muscular dystrophy is the most severe and common type. Cell-based therapy for muscular dystrophy has been under investigation for several decades, with a wide selection of cell types being studied, including tissue-specific stem cells and reprogrammed stem cells. Several immune-deficient mouse models of muscular dystrophy have been generated, in which human cells obtained from various sources are injected to assess their preclinical potential. After transplantation, the presence of engrafted human cells is detected via immunofluorescence staining, using antibodies that recognize human, but not mouse, proteins. Here we show that one antibody specific to human spectrin, which is commonly used to evaluate the efficacy of transplanted human cells in mouse muscle, detects myofibers in muscles of NOD/Rag1(null)mdx(5cv), NOD/LtSz-scid IL2Rγ(null) mice, or mdx nude mice, irrespective of whether they were injected with human cells. These "reactive" clusters are regenerating myofibers, which are normally present in dystrophic tissue and the spectrin antibody is likely recognizing utrophin, which contains spectrin-like repeats. Therefore, caution should be used in interpreting data based on detection of single human-specific proteins, and evaluation of human stem cell engraftment should be performed using multiple human-specific labeling strategies.
Abstract The tropomyosin genes ( TPM1-4 ) contribute to the functional diversity of skeletal muscle fibers. Since its discovery in 1988, the TPM3 gene has been recognized as an indispensable regulator of muscle contraction in slow muscle fibers. Recent advances suggest that TPM3 isoforms hold more extensive functions during skeletal muscle development and in postnatal muscle. Additionally, mutations in the TPM3 gene have been associated with the features of congenital myopathies. The use of different in vitro and in vivo model systems has leveraged the discovery of several disease mechanisms associated with TPM3-related myopathy. Yet, the precise mechanisms by which TPM3 mutations lead to muscle dysfunction remain unclear. This review consolidates over three decades of research about the role of TPM3 in skeletal muscle. Overall, the progress made has led to a better understanding of the phenotypic spectrum in patients affected by mutations in this gene. The comprehensive body of work generated over these decades has also laid robust groundwork for capturing the multiple functions this protein plays in muscle fibers.
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