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Terence A Partridge

Publications and source records attributed to Terence A Partridge.

28 records · Page 2Linked to original sources
PubMed2004

Optimisation of ultrasound-mediated gene transfer (sonoporation) in skeletal muscle cells.

Ultrasound (US) is a promising tool for facilitating direct gene transfer to skeletal muscle, but no systematic optimisation study has been performed. We exposed H2K myoblast cells to US with varying intensity of exposure and duration to evaluate its effect on cell viability and transfection efficiency using as endpoints transfection rate, average fluorescence intensity (fluorescence normalised by the number of transfected cells) and overall expression (the product of transfection rate and average fluorescence intensity) as indices. Cell viability decreased with exposure time and intensity, consistent with previous findings. Optimal setting of US was observed at the range of 0.5 to 1 W cm(-2) with duration of 20 s, producing maximum efficiency (transfection = 4.5%) in gene transfection with minimum cell toxicity (cell viability = 83%). Higher intensity alone or in combination with low intensity and long duration did not improve cell viability and transfection. The increase of eGFP (enhanced green fluorescence protein) plasmid concentration up to 200 microg per mL was related to an increase in average fluorescence intensity and overall expression. However, transfection rate saturated when DNA concentration reached 50 microg per mL despite initial increase with DNA concentration. The average fluorescence intensity was linearly proportional to the logarithm of DNA concentration, suggesting a diffusion-based model for DNA uptake under sonoporation. We conclude that low-intensity US irradiation provides a safe and effective alternative for gene delivery.

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PubMed2004

Stem cell therapies for neuromuscular diseases.

The promise of stem cells for therapy of a variety of disease conditions is one of the most heavily marketed commodities of modern biotechnology. Interest in this prospect has opened up and destabilized a large area of developmental biology, challenging some of its major tenets, and becoming a source of controversy. Much of the disagreement arises from apparently simple conflicts of findings, which may have as much to do with differences of interpretation as of actual data. However, outside a few favoured situations, the practical significance of most of the findings is marginal, with only trivial levels of stem cell participation in tissue reconstruction. Currently, much of the effort in stem cell research is devoted to identifying sources of stem cells and the diversity of fates they can be induced to adopt. Areas that are in need of development are those concerned with delivery to target tissues, improvement of conversion to the desired cell types and effectiveness of functional integration of these cells into the target tissue. Of these hurdles, perhaps the most important is the efficient delivery of stem cells to the appropriate sites. The most promising results in this respect have been achieved by either targeted injection to discrete sites or, for more diffuse targets, delivery via the intermediary of a bone marrow transplant. For the latter especially, we need a degree of improvement that can only come from a better understanding of the mechanisms involved.

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PubMed2003

Pax7 distribution in human skeletal muscle biopsies and myogenic tissue cultures.

Demonstration of the importance of the paired box transcription factor Pax7 for the murine myosatellite cell population, with persistent expression in mature skeletal muscle, prompted us to investigate the distribution of Pax7 protein in biopsy samples of normal and pathological human skeletal limb muscle. Immunostaining for M-cadherin, an adhesion molecule present at the interface between myofibre and satellite cell, and the characteristic position adjacent to the muscle fibre and beneath the fibre's basement membrane were used to identify satellite cells. Anti-Pax7 reactivity was found in the majority of satellite cells but a small population was Pax7 negative. Neither could we identify Pax7-positive nuclei in freshly regenerating myotubes or in presumed myoblasts in these biopsies. Similarly, in myogenic cell cultures derived from the explantation of human foetal muscle Pax7 expression was low or undetectable at the proliferative myoblast stage but it became prominent in an increasing proportion of mononucleate cells after the induction of differentiation. This expression was, however, restricted to mononucleate cells; it did not persist into the differentiation stage of newly formed multinucleate myotubes. Despite this, in the biopsy samples, we occasionally found Pax7-positive nuclei in muscle fibres that seemed to be undergoing degenerative changes. Most of these were found to be the nuclei of cells engaged in focal regenerative processes, but Pax7 re-expression by myonuclei "in distress" cannot be ruled out entirely.

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PubMed2003

Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse.

As a target for gene therapy, Duchenne muscular dystrophy (DMD) presents many obstacles but also an unparalleled prospect for correction by alternative splicing. The majority of mutations in the dystrophin gene occur in the region encoding the spectrin-like central rod domain, which is largely dispensable. Thus, splicing around mutations can generate a shortened but in-frame transcript, permitting translation of a partially functional dystrophin protein. We have tested this idea in vivo in the mdx dystrophic mouse (carrying a mutation in exon 23 of the dystrophin gene) by combining a potent transfection protocol with a 2-O-methylated phosphorothioated antisense oligoribonucleotide (2OMeAO) designed to promote skipping of the mutated exon*. The treated mice show persistent production of dystrophin at normal levels in large numbers of muscle fibers and show functional improvement of the treated muscle. Repeated administration enhances dystrophin expression without eliciting immune responses. Our data establishes the realistic practicality of an approach that is applicable, in principle, to a majority of cases of severe dystrophinopathy.

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PubMed2003

Stem cell route to neuromuscular therapies.

As applied to skeletal muscle, stem cell therapy is a reincarnation of myoblast transfer therapy that has resulted from recent advances in the cell biology of skeletal muscle. Both strategies envisage the reconstruction of damaged muscle from its precursors, but stem cell therapy employs precursors that are earlier in the developmental hierarchy. It is founded on demonstrations of apparently multipotential cells in a wide variety of tissues that can assume, among others, a myogenic phenotype. The main demonstrated advantage of such cells is that they are capable of colonizing many tissues, including skeletal and cardiac muscle via the blood vascular system, thereby providing the potential for a body-wide distribution of myogenic progenitors. From a practical viewpoint, the chief disadvantage is that such colonization has been many orders of magnitude too inefficient to be useful. Proposals for overcoming this drawback are the subject of much speculation but, so far, relatively little experimentation. This review attempts to give some perspective to the status of the stem cell as a therapeutic instrument for neuromuscular disease and to identify issues that need to be addressed for application of this technology.

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PubMed2003

Muscle satellite cells.

Skeletal muscle satellite cells are quiescent mononucleated myogenic cells, located between the sarcolemma and basement membrane of terminally-differentiated muscle fibres. These are normally quiescent in adult muscle, but act as a reserve population of cells, able to proliferate in response to injury and give rise to regenerated muscle and to more satellite cells. The recent discovery of a number of markers expressed by satellite cells has provided evidence that satellite cells, which had long been presumed to be a homogeneous population of muscle stem cells, may not be equivalent. It is possible that a sub-population of satellite cells may be derived from a more primitive stem cell. Satellite cell-derived muscle precursor cells may be used to repair and regenerate damaged or myopathic skeletal muscle, or to act as vectors for gene therapy. CELL FACTS: (1) Number of cells in body: 2 x 10(7) to 3 x 10(7) myonuclei/g, 20-25 kg muscle in average man; 2 x 10(5) to 10 x 10(5) satellite cells/g, i.e. approximately 1 x 10(10) to 2 x 10(10) satellite cells per person. (2) Main functions: repair and maintenance of skeletal muscle. (3) Turnover rate: close to zero in non-traumatic conditions-high in disease or severe trauma.

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PubMed2002

Kinetics of myoblast proliferation show that resident satellite cells are competent to fully regenerate skeletal muscle fibers.

The satellite cell compartment provides skeletal muscle with a remarkable capacity for regeneration. Here, we have used isolated myofibers to investigate the activation and proliferative potential of satellite cells. We have previously shown that satellite cells are heterogeneous: the majority express Myf5 and M-cadherin protein, presumably reflecting commitment to myogenesis, while a minority is negative for both. Although MyoD is rarely detected in quiescent satellite cells, over 98% of satellite cells contain MyoD within 24 h of stimulation. Significantly, MyoD is only observed in cells that are already expressing Myf5. In contrast, a minority population does not activate by the criteria of Myf5 or MyoD expression. Following the synchronous activation of the myogenic regulatory factor+ve satellite cells, their daughter myoblasts proliferate with a doubling time of approximately 17 h, irrespective of the fiber type (type I, IIa, or IIb) from which they originate. Although fast myofibers have fewer associated satellite cells than slow, and accordingly produce fewer myoblasts, each myofiber phenotype is associated with a complement of satellite cells that has sufficient proliferative potential to fully regenerate the parent myofiber within 4 days. This time course is similar to that observed in vivo following acute injury and indicates that cells other than satellite cells are not required for complete myofiber regeneration.

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PubMed2002

Myogenic cell proliferation and generation of a reversible tumorigenic phenotype are triggered by preirradiation of the recipient site.

Environmental influences have profound yet reversible effects on the behavior of resident cells. Earlier data have indicated that the amount of muscle formed from implanted myogenic cells is greatly augmented by prior irradiation (18 Gy) of the host mouse muscle. Here we confirm this phenomenon, showing that it varies between host mouse strains. However, it is unclear whether it is due to secretion of proliferative factors or reduction of antiproliferative agents. To investigate this further, we have exploited the observation that the immortal myogenic C2 C12 cell line forms tumors far more rapidly in irradiated than in nonirradiated host muscle. We show that the effect of preirradiation on tumor formation is persistent and dose dependent. However, C2 C12 cells are not irreversibly compelled to form undifferentiated tumor cells by the irradiated muscle environment and are still capable of forming large amounts of muscle when reimplanted into a nonirradiated muscle. In a clonal analysis of this effect, we discovered that C2 C12 cells have a bimodal propensity to form tumors; some clones form no tumors even after extensive periods in irradiated graft sites, whereas others rapidly form extensive tumors. This illustrates the subtle interplay between the phenotype of implanted cells and the factors in the muscle environment.

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PubMed2002

Single fibers of skeletal muscle as a novel graft for cell transplantation to the heart.

OBJECTIVE: Skeletal myoblast transplantation is a promising alternative to treat heart failure. A single fiber, the minimal functional unit of skeletal muscle, retains skeletal myoblasts beneath the basal lamina. When surrounding muscle is injured, myoblasts migrate from the fiber into the damaged area to regenerate muscle. We hypothesized that such isolated fibers could be used as an efficient vehicle to deliver myoblasts into damaged myocardium, resulting in improved cardiac function. METHODS: Living single fibers of rat skeletal muscle were isolated, and their behavior was characterized in vitro. Single fibers were injected into the myocardium (at 4 sites, each receiving a single fiber) of rats in 2 models of heart failure induced either by means of doxorubicin administration or left coronary artery occlusion. RESULTS: Skeletal myoblasts dissociated from an isolated single fiber, proliferated, and differentiated into multinucleated myotubes in vitro. Within 3 days after grafting in vivo, original fibers provided putative myoblasts and disappeared. At 4 weeks, discrete loci consisting of several multinucleated myotubes were observed. Furthermore, single-fiber transplantation significantly improved cardiac function compared with the control treatment in either doxorubicin-treated hearts (maximum dP/dt, 4013.9 +/- 96.1 vs 3603.1 +/- 102.3 mm Hg/s; minimum dP/dt, -2313.7 +/- 75.1 vs. -2057.1 +/- 52.4 mm Hg/s) or ischemic hearts (maximum dP/dt, 3905.6 +/- 103.0 vs 3572.6 +/- 109.7 mm Hg/s; minimum dP/dt, -2336.1 +/- 69.7 vs -2106.4 +/- 74.2 mm Hg/s). CONCLUSION: Single-fiber transplantation acts as a vehicle for delivering putative skeletal myoblasts that appear to differentiate into myotubes within the myocardium. This was associated with improved function of failing hearts, suggesting its efficacy as a novel graft for cellular cardiomyoplasty.

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