Paul C. Holland

Bio: Paul C. Holland is an academic researcher from Montreal Neurological Institute and Hospital. The author has contributed to research in topics: Skeletal muscle & Myocyte. The author has an hindex of 28, co-authored 47 publications receiving 2668 citations. Previous affiliations of Paul C. Holland include McGill University.

Myoblast transfer in duchenne muscular dystrophy

Abstract: One biceps muscle of 8 patients with Duchenne muscular dystrophy was injected at 55 sites with a total of 55 million viable, purified, and contamination-free normal myoblasts (myoblast transfer). The other biceps of each patient was injected with a placebo to serve as a control. The procedure was blinded to the patients, parents, and investigators. Myoblasts derived from a biopsy specimen of the fathers were cultured and purified under strict conditions and carefully screened for microbial contamination. All patients received cyclophosphamide for immunosuppression for 6 or 12 months. No serious complications were observed after myoblast transfer, indicating that the procedure is safe. The overall therapeutic efficiency of myoblast transfer was poor as judged by the results in maximal voluntary force generation, dystrophin content of the muscle, magnetic resonance imaging of the muscle, and the lack of donor-derived DNA and dystrophin messenger RNA in the injected muscle. An improved efficiency of the take of myoblasts might be achieved by using younger cells and injecting the myoblasts with a myonecrotic agent (to increase the prevalence of regeneration) and a basal laminal fenestrating agent.

Dystrophin is expressed in mdx skeletal muscle fibers after normal myoblast implantation.

Abstract: In mdx mice, the dystrophin gene of the X chromosome is defective and, as a result, immunoreactive dystrophin is undetectable in all muscle fibers of all animals of this highly inbred strain. This study showed that implantation of suspensions of clonal cultures of normal human myoblasts into different regions of quadriceps muscles of 6-to-10-day-old mdx mice or 60-day-old mdx mice (whose muscles have been crushed 4 days before implantation) results in the appearance of scattered fiber segments containing microscopically demonstrable immunoreactive dystrophin. In the animals that received the normal myoblast implantation in the prenecrotic stage of the disease (6 to 10 days of age), the dystrophin-positive fiber segments (demonstrated at ages 35, 45, and 60 days) escaped necrosis. This was determined by the absence of the characteristic chains of central nuclei, a reliable marker of prior necrosis in mdx muscle fibers. By heavy labeling of the nuclear DNA of the transplantable human myoblasts with H3-thymidine during culturing, and by sequential performance of an immunocytochemical staining for dystrophin and autoradiography on the same sections, some dystrophin-positive fiber segments were shown to contain radiolabeled myonuclei. It was concluded that nondystrophic myoblasts fused with host muscle fibers to form mosaic muscle fibers in which the normal dystrophin gene of the implanted myoblasts was expressed. This approach may be employed for the mitigation of the deleterious consequences of a gene defect in recessively inherited human muscle diseases such as Duchenne dystrophy.

A differential efficiency of adenovirus-mediated in vivo gene transfer into skeletal muscle cells of different maturity

Abstract: High titre (10(11)-10(12) pfu/ml) suspensions of autonomously replication-defective type 5 human adenovirus (AV) recombinants with different reporter gene inserts (CMV-Luciferase (Lux), CMV-beta-galactosidase (Lac Z), RSV-Lux and RSV-Lac Z) were injected into intact quadriceps muscles of 1-5 day old (Group 1) or 35-45 day old (Group 2) normal mice, as well as regenerating adult mouse muscles (Group 3) and 35 day old mdx muscles (Group 4). The expression of the reporter genes was quantitated 10 days and 2 months later. At 10 days postinjection all reporter gene expression was very high in the neonatally injected (Group 1) muscles. In Group 2 muscles the transduction was markedly less. In Group 3 muscles the gene expression was significantly better than in the Group 2 muscles. In adult mdx muscles (Group 4) where spontaneous regeneration is usually present, the results were similar to those in Group 3 animals. At 2 months post-injection in Group 1 animals, the RSV-Lux expression was even higher than at 10 days postinjection. The cell surface density of alpha v-integrin-containing molecules including the internalization receptor for AV in Groups 1, 2, 3 and 4 showed a positive correlation with AV transducibility. We conclude that adenovirus vector in high titre (10(10) pfu/ml or above) is capable of efficiently transducing only immature muscle cells but not mature muscle fibers in vivo and this appears to correlate with a higher surface density of the available AV internalization receptor in immature muscle cells and lower level in mature muscle fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the primary coxsackie and adenovirus receptor is downregulated during skeletal muscle maturation and limits the efficacy of adenovirus-mediated gene delivery to muscle cells.

Abstract: Skeletal muscle fibers are infected efficiently by adenoviral vectors only in neonatal animals. This lack of tropism for mature skeletal muscle may be partly due to inefficient binding of adenoviral particles to the cell surface. We evaluated in developing mouse muscle the expression levels of two high-affinity receptors for adenovirus, MHC class I and the coxsackie and adenovirus receptor (CAR). The moderate levels of MHC class I transcripts that were detected in quadriceps, gastrocnemius, and heart muscle did not vary between postnatal day 3 and day 60 adult tissue. A low level of CAR expression was detected on postnatal day 3 in quadriceps and gastrocnemius muscles, but CAR expression was barely detectable in adult skeletal muscle even by reverse transcriptase-polymerase chain reaction. In contrast, CAR transcripts were moderately abundant at all stages of heart muscle development. Ectopic expression of CAR in C2C12 mouse myoblast cells increased their transducibility by adenovirus at all multiplicities of infection (MOIs) tested as measured by lacZ reporter gene activity following AVCMVlacZ infection, with an 80-fold difference between CAR-expressing cells and control C2C12 cells at an MOI of 50. Primary myoblasts ectopically expressing CAR were injected into muscles of syngeneic hosts; following incorporation of the exogenous myoblasts into host myofibers, an increased transducibility of adult muscle fibers by AVCMVlacZ was observed in the host. Expression of the lacZ reporter gene in host myofibers coincided with CAR immunoreactivity. Furthermore, sarcolemmal CAR expression was markedly increased in regenerating muscle fibers of the dystrophic mdx mouse, fibers that are susceptible to adenovirus transduction. These analyses show that CAR expression by skeletal muscle correlates with its susceptibility to adenovirus transduction, and that forced CAR expression in mature myofibers dramatically increases their susceptibility to adenovirus transduction.

Localization and quantitation of the chromosome 6-encoded dystrophin-related protein in normal and pathological human muscle.

Abstract: A dystrophin-related protein (DRP) encoded by a gene on chromosome 6 was studied in 14 normal and 79 pathological human skeletal muscle biopsies, as well as in cultured myotubes by light microscopic immunocytochemistry and quantitative immunoblots. In normal muscle immunoreactive DRP was present at the postjunctional surface membrane, at the surface of satellite cells, in the walls of blood vessels, in Schwann cells and in perineurium of intramuscular nerves. All of this produced a weak signal on immunoblots. In Duchenne/Becker dystrophy (DMD/BMD) and in polymyositis (PM) or dermatomyositis (DM) DRP was present throughout the extrajunctional surface membrane of extra- and intrafusal muscle fibers, particularly regenerating ones. This produced a 15–17-fold increase of DRP over normal in DMD/BMD and 4–10-fold increase over normal in PM and DM on immunoblots. In other pathological muscles, DRP localization pattern and quantity was about the same as in normals. Dystrophin-related protein was present in about the same amounts and distribution in normal and DMD cultured myoblasts and myotubes. The molecular stimulus for the marked upregulation of DRP in DMD/BMD and in the inflammatory myopathies is not known. In DMD/BMD the diffuse sarcolemmal DRP may partially compensate for dystrophin deficiency.

Fiber types in mammalian skeletal muscles.

Abstract: Mammalian skeletal muscle comprises different fiber types, whose identity is first established during embryonic development by intrinsic myogenic control mechanisms and is later modulated by neural and hormonal factors. The relative proportion of the different fiber types varies strikingly between species, and in humans shows significant variability between individuals. Myosin heavy chain isoforms, whose complete inventory and expression pattern are now available, provide a useful marker for fiber types, both for the four major forms present in trunk and limb muscles and the minor forms present in head and neck muscles. However, muscle fiber diversity involves all functional muscle cell compartments, including membrane excitation, excitation-contraction coupling, contractile machinery, cytoskeleton scaffold, and energy supply systems. Variations within each compartment are limited by the need of matching fiber type properties between different compartments. Nerve activity is a major control mechanism of the fiber type profile, and multiple signaling pathways are implicated in activity-dependent changes of muscle fibers. The characterization of these pathways is raising increasing interest in clinical medicine, given the potentially beneficial effects of muscle fiber type switching in the prevention and treatment of metabolic diseases.

Dystrophin expression in the mdx mouse restored by stem cell transplantation

Abstract: The development of cell or gene therapies for diseases involving cells that are widely distributed throughout the body has been severely hampered by the inability to achieve the disseminated delivery of cells or genes to the affected tissues or organ. Here we report the results of bone marrow transplantation studies in the mdx mouse, an animal model of Duchenne's muscular dystrophy, which indicate that the intravenous injection of either normal haematopoietic stem cells or a novel population of muscle-derived stem cells into irradiated animals results in the reconstitution of the haematopoietic compartment of the transplanted recipients, the incorporation of donor-derived nuclei into muscle, and the partial restoration of dystrophin expression in the affected muscle. These results suggest that the transplantation of different stem cell populations, using the procedures of bone marrow transplantation, might provide an unanticipated avenue for treating muscular dystrophy as well as other diseases where the systemic delivery of therapeutic cells to sites throughout the body is critical. Our studies also suggest that the inherent developmental potential of stem cells isolated from diverse tissues or organs may be more similar than previously anticipated.

Function and genetics of dystrophin and dystrophin-related proteins in muscle

Abstract: The X-linked muscle-wasting disease Duchenne muscular dystrophy is caused by mutations in the gene encoding dystrophin. There is currently no effective treatment for the disease; however, the complex molecular pathology of this disorder is now being unravelled. Dystrophin is located at the muscle sarcolemma in a membrane-spanning protein complex that connects the cytoskeleton to the basal lamina. Mutations in many components of the dystrophin protein complex cause other forms of autosomally inherited muscular dystrophy, indicating the importance of this complex in normal muscle function. Although the precise function of dystrophin is unknown, the lack of protein causes membrane destabilization and the activation of multiple pathophysiological processes, many of which converge on alterations in intracellular calcium handling. Dystrophin is also the prototype of a family of dystrophin-related proteins, many of which are found in muscle. This family includes utrophin and α-dystrobrevin, which are involved in the maintenance of the neuromuscular junction architecture and in muscle homeostasis. New insights into the pathophysiology of dystrophic muscle, the identification of compensating proteins, and the discovery of new binding partners are paving the way for novel therapeutic strategies to treat this fatal muscle disease. This review discusses the role of the dystrophin complex and protein family in muscle and describes the physiological processes that are affected in Duchenne muscular dystrophy.

An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control

Abstract: BACKGROUND In adult mammalian organisms, multiple tissues—including the skin, blood, stomach, and intestines—are entrapped in a state of permanent regeneration; older cells are constantly shed, and the tissue is continuously being regenerated from resident stem cells. This phenomenon of “tissue renewal” was appreciated by Leblond in 1956, but the underlying mechanism has been unclear. It is now evident that a class of extracellular developmental signaling proteins, known as Wnt signals, animate the continued renewal of several mammalian tissues by fuelling stem cell activity. If the Wnt pathway is inhibited, tissue renewal is crippled. This signaling pathway is an ancient evolutionary program dating from when Wnt signals arose in the simplest multicellular organisms, in which Wnts acted as primordial symmetry-breaking signals crucial for the generation of patterned tissues during embryogenesis. In vertebrates, these signals also function in pattern maintenance: They sustain tissue renewal, enabling tissues to be continuously replenished and maintained over a lifetime. Multiple adult organs are in a state of continual regeneration. In tissues such as the skin, intestines, brain, and mammary glands, Wnt signaling proteins sustain this constant regeneration by inducing stem cells (green cells in the illustration) to grow. This leads to the robust supply of new cells (green) in order to replenish and maintain the tissue. [Image credits are available in the full article online.] ADVANCES In contrast to traditional “long-range” developmental signals, Wnts seem to act as short-range intercellular signals—acting mostly between adjacent cells. Lending credence to this notion, a membrane-tethered Wnt protein variant can fulfill most functions of a normal Wnt protein in Drosophila . Likely explaining the short-range nature of these signals, Wnt proteins are attached to a lipid and therefore are hydrophobic; they cannot freely traverse the extracellular space by themselves. This provides insight into how tissue renewal is regulated. It implies that Wnt signals emanating from the stem cell microenvironment (the “niche”) may influence adjacent stem cells without affecting a broad field of cells located farther away. The concept of an external niche, however, may have to be refined because it is clear that stem cells can sometimes act as their own niche and have unexpected developmental self-organizing capacities. Last, the widespread importance of Wnt signaling in driving tissue renewal has been revealed by the identification of Axin2 and Lgr5 , genes expressed in cells that are responding to Wnt signals. Genetically labeling Axin2 + or Lgr5 + cells in a variety of tissues has revealed that such cells fuel tissue renewal in the intestines, mammary gland, skin, and brain, among other organs. OUTLOOK The amazing continuous self-regeneration of various mammalian tissues over years and decades continues to be an enigmatic terra incognita in biology. For instance, visualization of stem cells in real-time in vivo (through intravital microscopy) has shown that when some stem cells are ablated, they are replaced by more differentiated cells that are recalled to the stem cell niche, whereupon they regain stem cell identity to effect tissue repair. Therefore, lineage barriers between stem cell and differentiated fates are not always stringent and can be traversed during times of tissue damage. Reactivated Wnt signals may be instrumental in this process, and perhaps such signals could be exploited in order to enkindle tissue regeneration after injury or disease. From a pragmatic perspective, Wnt signals have already found practical use in manipulating stem cells, enabling propagation of stem cells in vitro as self-renewing cell populations and as organoids.

Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.

Abstract: The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity--suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.