A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use.

Autologous keratinocytes can be used to augment cutaneous repair, such as in the treatment of severe burns and recalcitrant ulcers. Such cells can be delivered to the wound bed either as a confluent sheet of cells or in single-cell suspension. The standard method for expanding primary human keratinocytes in culture uses lethally irradiated mouse 3T3 fibroblasts as feeder cells to support keratinocyte attachment and growth. In an effort to eliminate xenobiotic cells from clinical culture protocols where keratinocytes are applied to patients, we investigated whether human autologous primary fibroblasts could be used to expand keratinocytes in culture. At a defined ratio of a 6:1 excess of keratinocytes to fibroblasts, this co-culture method displayed a population doubling rate comparable to culture with lethally irradiated 3T3 cells. Furthermore, morphological and molecular analysis showed that human keratinocytes expanded in co-culture with autologous human fibroblasts were positive for proliferation markers and negative for differentiation markers. Keratinocytes expanded by this method thus retain their proliferative phenotype, an important feature in enhancing rapid wound closure. We suggest that this novel co-culture method is therefore suitable for clinical use as it dispenses with the need for lethally irradiated 3T3 cells in the rapid expansion of autologous human keratinocytes.

Substrate-induced phenotypical change of monocytes/macrophages into myofibroblast-like cells: a new insight into the mechanism of in-stent restenosis.

Stented coronary angioplasty is the procedure of choice to re-establish patency in obstructed coronary arteries. However, the stent implantation procedure often leads to in-stent restenosis, a process that is characterized by stent strut colonization by macrophages and smooth muscle cells and by neointima formation. The present in vitro study investigates the effect of stent materials on the phenotypical features of monocyte/macrophages. Human peripheral blood monocytes from healthy donors (n = 7) were cultured up to 7 days on substrates mimicking: (i) the stent surface (i.e., electropolished stainless steel), (ii) the de-endothelialized vessel wall (collagen-based extracellular matrix gel), and (iii) thrombus (i.e., fibrin gel). The cells were analyzed by immunocytochemistry for their ability to express alpha-actin, a typical myofibroblast marker, by ELISA to determine PDGF-BB and TGF-beta1 secretion and by PCR to evaluate hyaluronan synthase 1, 2, and 3 genes expression. Data were statistically analyzed by ANOVA (Dunnett's test) and data considered significantly different at p

The internervous safe zone for incision of the capsule of the hip. A cadaver study.

In order to determine the potential for an internervous safe zone, 20 hips from human cadavers were dissected to map out the precise pattern of innervation of the hip capsule. The results were illustrated in the form of a clock face. The reference point for measurement was the inferior acetabular notch, representing six o'clock. Capsular branches from between five and seven nerves contributed to each hip joint, and were found to innervate the capsule in a relatively constant pattern. An internervous safe zone was identified anterosuperiorly in an arc of 45 degrees between the positions of one o'clock and half past two. Our study shows that there is an internervous zone that could be safely used in a capsule-retaining anterior, anterolateral or lateral approach to the hip, or during portal placement in hip arthroscopy.

Radial nerve contribution to brachialis in the UK Caucasian population: position is predictable based on surface landmarks.

The midline of the brachialis muscle is now regarded by many surgeons as an internervous plane, between the musculocutaneous nerve supply to the medial portion and a radial nerve contribution to the lateral portion, a principle applied in anterior approaches to the humerus. The radial nerve to brachialis has been described previously in East Asian specimens (Ip and Chang, 1968, Anat Rec 162:363-371; Mahakkanukrauh and Somsarp, 2002, Clin Anat 15:206-209). We sought to investigate the occurrence and character of this nerve in the UK Caucasian population. Dissection of the musculocutaneous nerve and radial nerve was carried out in Caucasian cadaveric arms (n = 42). The radial nerve was dissected from the spiral groove to the lateral epicondyle. Where a branch to brachialis was encountered, its course and level of origin relative to the length of the arm were noted. The musculocutaneous nerve innervated the brachialis in all specimens. Upon investigation 67% of specimens were found to have a radial nerve branch to brachialis. Sixty-one percent of these branches went straight into the muscle, 13% descended, and 26% recurred. The level of origin of the radial nerve branch to brachialis was at a mean of 23% of the distance between the lateral epicondyle and the acromion (n = 31, range 17-37%, SD = 5.3). In three specimens, two branches were observed. A radial nerve contribution to the innervation of the brachialis was present in 67% of UK Caucasian cadavers, which is less than that noted by others in East Asian specimens. The level of the origin of these branches is predictable based on surface landmarks. This fact may be of use during humeral surgery.

The muscle-specific marker desmin is expressed in a proportion of human dermal fibroblasts after their exposure to galectin-1.

We have previously shown that galectin-1 is a factor capable of converting mouse dermal fibroblasts to the myogenic lineage [Cell Transplant 2000;9:519]. Here, we report that human dermal fibroblasts are also capable of expressing the myogenic marker, desmin, when grown in muscle-cell-conditioned media. Furthermore, the human foetal skin cells also express this marker when grown in the presence of galectin-1. These results highlight the importance of galectin-1 in the conversion of both human and murine skin cells to a myogenic lineage. Thus galectin-1 could be an important tool for use in autologous cell therapies for the treatment of human muscular dystrophies.

Dermal fibroblasts participate in the formation of new muscle fibres when implanted into regenerating normal mouse muscle.

Both in vitro and in vivo studies have described the conversion of fibroblasts to myogenesis when in the presence of dysfunctional myogenic cells. Myogenic conversion of fibroblasts subjected to a normal, as opposed to a diseased muscle environment has only been reported in vitro. The primary aim of this work was to determine if fibroblasts can convert to a myogenic lineage and contribute to new fibre formation when implanted into the regenerating muscle of a normal mouse. Dermal fibroblasts were prepared from neonatal mouse skin and labelled prior to implantation with the fluorescent nuclear marker 4',6-diamidino-2-phenylindole (DAPI). Cells were implanted into muscles of host mice that had been subjected to either cold/crush or minced muscle injury. Some host muscles were x-irradiated to deplete the muscle of endogenous muscle precursor cells. Muscles were removed at 3 wk postimplantation and analysed both histologically and for the presence of DAPI labelled nuclei. Fibres containing DAPI labelled central nuclei indicated that the implanted cells had participated in the regenerative process. Mouse dermal fibroblasts therefore do contribute to muscle fibre formation in regenerating normal mouse muscle but the extent of their contribution is dependent on the nature of the trauma induced in the host muscle. The study also showed that regeneration was more successful in muscles which had not been irradiated, which is contrary to the previous studies where dermal fibroblasts were introduced into myopathic mouse muscle.

A factor implicated in the myogenic conversion of nonmuscle cells derived from the mouse dermis.

Using the mdx mouse model for human Duchenne muscular dystrophy we have shown that a cell population residing in the dermis of C57B1/10ScSn mouse skin is capable of converting to a myogenic lineage when implanted into the mdx muscle environment. It was important to determine the characteristics of the converting cell. A previous in vitro study indicated that 10% of cells underwent conversion but only when the cells were grown in medium previously harvested from a myogenic culture. In the present study we cloned cells derived from the dermis to identify the converting cells. Clones grown in normal growth medium showed no conversion, but when grown in medium conditioned by muscle cells around 40% conversion was achieved in several individual clones. We investigated whether the protein beta-galactoside binding protein (betaGBP), which is secreted by myoblasts and acts as a cell growth regulator of fibroblasts. could be a candidate factor responsible for conversion. Medium harvested from COS-1 cells infected with a construct containing betaGBP has been used for this investigation. Growth of dermal fibroblasts in medium enriched with this factor showed a high rate of conversion to cells expressing muscle-specific factors.

Independent regulation of the myotonic dystrophy 1 locus genes postnatally and during adult skeletal muscle regeneration.

Myotonic dystrophy is caused by a CTG(n) expansion in the 3'-untranslated region of a serine/threonine protein kinase gene (DMPK), which is flanked by two other genes, DMWD and SIX5. One hypothesis to explain the wide-ranging effects of this expansion is that, as the mutation expands, it alters the expression of one or more of these genes. The effects may vary in different tissues and developmental stages, but it has been difficult to develop these hypotheses as the normal postnatal developmental expression patterns of these genes have not been adequately investigated. We have developed accurate transcript quantification based on fluorescent real-time reverse transcription-polymerase chain reaction (TaqMan) to develop gene expression profiles during postnatal development in C57Bl/10 mice. Our results show extensive independent postnatal regulation of the myotonic dystrophy-locus genes in selected tissues and demonstrate which are the most highly expressed of the genes in each tissue. All three genes at the locus are expressed in the adult lens, questioning a previous model of cataractogenesis mediated solely by effects on Six5 expression. Additionally, using an in vivo model, we have shown that Dmpk levels decrease during the early stages of muscle regeneration. Our data provide a framework for investigation of tissue-specific pathological mechanisms in this disorder.

Transgenic mice ubiquitously expressing human placental alkaline phosphatase (PLAP): an additional reporter gene for use in tandem with beta-galactosidase (lacZ).

A fundamental keystone of developmental biology has been the growing use of reporter genes in model transgenic systems. Their use has greatly facilitated investigations of cell lineage and cell fate in addition to aiding experiments aimed at determining patterns of gene expression, gene interaction and gene regulation. Through construction of transgenic mice, ubiquitously expressing human placental alkaline phosphatase (PLAP), we demonstrate the suitability of PLAP as a reporter gene for use in conjunction with, or as an alternative to, beta-galactosidase (lacZ). Our findings demonstrate that over-expression of PLAP has no adverse effects on mouse development or viability, despite a widespread pattern of expression. This technology provides a simple yet effective mechanism based on eukaryotic reporter gene technology to facilitate the identification of transgenic cells within complex in vivo systems.

SCID mice containing muscle with human mitochondrial DNA mutations. An animal model for mitochondrial DNA defects.

Defects of the mitochondrial genome are important causes of disease. Despite major advances in our investigation of patients, there is no effective therapy. Progress in this area is limited by the absence of any animal models in which we can evaluate treatment. To develop such a model we have injected human myoblasts into the tibialis anterior of SCID mice after inducing necrosis. After injection of normal human myoblasts, regenerating fibers expressed human beta-spectrin, confirming they were derived from fusion of human myoblasts. The stability of the muscle fibers was inferred by demonstrating the formation of motor end plates on the regenerating fibers. In addition, we show the presence of human cytochrome c oxidase subunit II, which is encoded by the mitochondrial genome, in the regenerated fibers. After injection of human myoblasts containing either the A8344G or the T8993C heteroplasmic mitochondrial DNA mutations, human beta-spectrin positive fibers were found to contain the mutation at a similar level to the injected myoblasts. These studies highlight the potential value of this model for the study of mitochondrial DNA defects.

Over expression of the murine myotonic dystrophy protein kinase in the mouse myogenic C2C12 cell line leads to inhibition of terminal differentiation.

Myotonic dystrophy (DM) is an autosomal dominant human disorder, caused by the abnormal expansion of a CTG trinucleotide repeat in the 3' untranslated region of a protein kinase gene (DMPK). Muscle symptoms are a common feature of the disorder and in the adult onset cases there are increased patterns of muscle fibre degeneration and regeneration. In the congenitally affected infants there is a failure of muscle maturation, with the histological presence of numerous immature fibres. However, the pathological mechanism in both forms of the disease is unclear. We report that over-expression of the murine dmpk gene, in a murine myogenic cell line, leads to markedly reduced levels of fusion to the terminally differentiated state. These findings complement recently published data using a heterologous expression/cell system and may have implications for the understanding of the disease process in this disorder.

Differential labelling of laminin alpha 2 in muscle and neural tissue of dy/dy mice: are there isoforms of the laminin alpha 2 chain?

Laminin alpha 2, a sub-unit of the basement membrane component laminin-2, is deficient in the dy/dy and allelic dy2 J/dy2 J mouse. It is also the defective protein in a proportion of children with congenital muscular dystrophy. Linkage and mutational analysis have established that this is a primary effect caused by defects in the LAMA2 gene. Laminin alpha 2 has previously been shown to be deficient in dy/dy skeletal muscle, peripheral nervous system and brain. We report here preliminary observations on differences in detection of laminin alpha 2 in muscle, peripheral nerves and brain of dy/dy mice using three, well characterized antibodies. In normal muscle laminin alpha 2 is localized to the basement membrane of the myofibres and the Schwann cells of peripheral nerves, whilst in adult brain it is only detected on blood vessels. Our results show that there is appreciable, but slightly reduced, expression of laminin alpha 2 in skeletal muscle of dy/dy mice but almost no detectable protein in the brain, peripheral nerve and spinal nerve roots. Our observations are at present unexplained but they raise the possibility for the first time that there may be different tissue specific isoforms of laminin alpha 2. Molecular characterization of possible differences responsible for our observations may aid the identification of the mutation in the dy/dy mouse and lead to a better understanding of the role and expression of laminin alpha 2 in pathological conditions.

Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy.

The absence of dystrophin at the muscle membrane leads to Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease that is inevitably fatal in early adulthood. In contrast, dystrophin-deficient mdx mice appear physically normal despite their underlying muscle pathology. We describe mice deficient for both dystrophin and the dystrophin-related protein utrophin. These mice show many signs typical of DMD in humans: they show severe progressive muscular dystrophy that results in premature death, they have ultrastructural neuromuscular and myotendinous junction abnormalities, and they aberrantly coexpress myosin heavy chain isoforms within a fiber. The data suggest that utrophin and dystrophin have complementing roles in normal functional or developmental pathways in muscle. Detailed study of these mice should provide novel insights into the pathogenesis of DMD and provide an improved model for rapid evaluation of gene therapy strategies.

Conversion of dermal fibroblasts to a myogenic lineage is induced by a soluble factor derived from myoblasts.

The limb and axial skeletal muscles of mammals originate from somitic dermomyotome, which during early development separates to form two discrete structures, the dermatome and the myotome. The latter cell mass gives rise to the muscle-forming lineage while cells of the dermatome will form the skin dermal fibroblast population of the dorsal regions of the body. It has been generally accepted for some time that myotome-derived myoblasts were the sole source of muscle fibre nuclei, but evidence has recently been presented from several laboratories that fibroblasts can fuse with myoblasts to contribute active nuclei to the resulting myotubes. We report here an investigation into the myogenic capacity of fibroblasts. Confluent monocultures of mouse dermal fibroblasts, muscle fibroblasts, and C2C12 myoblasts each retain their individual phenotype when maintained for periods up to 7 days in culture. We also grew isolated colonies of fibroblasts and myoblasts in an arrangement which allowed free exchange of tissue culture medium between the 2 cell types. We found evidence of the conversion of dermal fibroblasts to a myogenic lineage as measured by the appearance of MyoD-positive cells expressing the muscle-specific intermediate filament desmin. In addition, dermal fibroblast cultures contained multinucleate syncytia positive for MyoD and containing sarcomeric myosin heavy chain. In contrast, muscle-derived fibroblasts showed no evidence of myogenic conversion when maintained in identical culture conditions. We prepared conditioned medium from confluent cultures of C2C12 myoblasts and added this material to confluent monocultures of either dermal or muscle fibroblasts. While muscle fibroblasts showed no phenotypic alterations, cultures of dermal fibroblasts responded to myoblast conditioned medium by converting to a myogenic lineage as judged by expression of MyoD and desmin. We conclude that a proportion of dermal fibroblasts retain a myogenic capacity into stages well beyond their early association with myoblasts in the dermomyotome.

Dermal fibroblasts convert to a myogenic lineage in mdx mouse muscle.

Duchenne muscular dystrophy is a primary muscle disease that manifests itself in young boys as a result of a defect in a gene located on the X-chromosome. This gene codes for dystrophin, a normal muscle protein that is located beneath the sarcolemma of muscle fibres. Therapies to alleviate this disease have centred on implanting normal muscle precursor cells into dystrophic fibres to compensate for the lack of this gene and its product. To date, donor cells for implantation in such therapy have been of myogenic origin, derived from paternal biopsies. Success in human muscle, however, has been limited and may reflect immune rejection problems. To overcome this problem the patient's own myogenic cells, with the dystrophin gene inserted, could be used, but this could lead to other problems, since these cells are those that are functionally compromised by the disease. Here, we report the presence of high numbers of dystrophin-positive fibres after implanting dermal fibroblasts from normal mice into the muscle of the mdx mouse-the genetic homologue of Duchenne muscular dystrophy. Dystrophin-positive fibres were also abundant in mdx muscle following the implantation of cloned dermal fibroblasts from the normal mouse. Our results suggest the in vivo conversion of these non-myogenic cells to the myogenic pathway resulting in the formation of dystrophin-positive muscle fibres in the deficient host. The use of dermal fibroblasts may provide an alternative approach to the previously attempted myoblast transfer therapy, which in human trials has yielded disappointing results.

Myoblasts fail to stimulate T cells but induce tolerance.

Recent interest in myoblast transfer and in the use of myoblasts as vehicles in gene therapy has made it important to understand the potential immunogenicity of allogeneic or neoantigen-expressing myoblasts. Given the problems of producing a pure population of myoblasts, in this study we used a tumour-derived muscle cell line (TE671), with phenotypic features of myoblasts, which we transfected to express HLA-DR1. However, this cell line was unable to stimulate either established HLA-DR1-specific allorective T cell clones or a primary alloresponse. Nor could it present haemagglutinin peptide HA 306-324 to DR1-restricted, HA 306-324-specific T cell clones or lines. Indeed, preincubation with DR1-expressing TE671 and HA 306-324 rendered such T cells tolerant as judged by their subsequent inability to proliferate in response to a DR1+ B cell line plus peptide HA 306-324. These results imply that myoblasts do not provide costimulatory signals, and are therefore unlikely to stimulate allospecific T cells following myoblasts transplantation or to initiate neoantigen-specific immune responses following in vivo transfection.

Magnetic affinity cell sorting (MACS) separation and flow cytometric characterization of neural cell adhesion molecule-positive, cultured myogenic cells from normal and dystrophic dogs.

We developed a magnetic affinity cell sorting (MACS) assay based on differential expression of neural cell adhesion molecule (NCAM) isoforms in muscle cell cultures from normal and dystrophic dogs. NCAM is expressed during normal muscle differentiation, but has not been extensively examined within the context of muscle disease. A myogenic MACS assay could potentially maximize chances of obtaining normal nonsenescent, low-passage myogenic cells capable of proliferating in vitro and in vivo following transplantation. Myoblast-specific anti-NCAM polyclonal antibody directed against the NCAM isoform associated with muscle cell proliferation more effectively separated mixed canine cultures than did monoclonal antibodies directed against differentiated NCAM isoforms in the MACS assay. Flow cytometry using 5.1H11 anti-NCAM monoclonal antibody was then performed on normal and dystrophic fractionated cells and the results from these two groups were compared to each other and to nonfractionated cell populations. Normal canine cell cultures that had not been separated contained a larger percentage of FACscan-positive cells than did corresponding dystrophic canine cell cultures. Prior polyclonal anti-NCAM MACS separation of dystrophic cultures yielded higher numbers of adherent cells and higher gating percentages of 5.1H11-positive cell populations than did normal cultures. However, cells from dystrophic animals exhibited lower mean fluorescent expression of NCAM than normal cells.

Migration of lacZ positive cells from the tibialis anterior to the extensor digitorum longus muscle of the X-linked muscular dystrophic (mdx) mouse.

C2 mouse myogenic cells carrying the lacZ gene coding for beta-galactosidase (beta-gal) were injected into the tibialis anterior muscle of dystrophin-deficient mdx mice. Introduced cells were shown to have been incorporated into fibres of the injected muscle by virtue of the colocalization of beta-gal and dystrophin within them. Synthetic Nuclepore membrane inserted between the injected tibialis anterior and adjacent extensor digitorum longus muscle permitted the visualization of cells migrating between the two muscles through the pores of the membrane. Although the exact nature of the cells passing through the Nuclepore could not be determined by this method, they were thought to include implanted myogenic cells. Evidence for this was gained by the presence of beta-gal/dystrophin positive fibres within the extensor digitorum longus. Incorporation of cells into the adjacent extensor digitorum longus was greater in animals where this muscle had been autografted by the cutting and resuturing of the distal tendon. Autografted extensor digitorum longi differed from those which had not been subject to this procedure, by undergoing extensive fibre degeneration followed by regeneration, and further by the stripping of their surrounding epimysial covering. Implanted cells substantially participated in extensor digitorum longus fibre formation in these mice, up to 31% of their fibres 3 weeks after implantation coexpressing both the introduced lacZ gene product and the dystrophin gene product, the latter not normally expressed within the fibres of this myopathic recipient.