Abstract Myoblast migration requires matrix metalloproteinase (MMP) activity but the contribution of individual MMPs or tissue inhibitors of matrix metalloproteinase (TIMPs), particularly MMP‐9 and TIMP‐1, is lacking. Using two clones derived for differential regulation of MMP‐2, MMP‐9, and TIMP‐1, we correlated protein expression with cell migration. MMP/TIMP regulation was determined by zymography, western blots, and quantitative reverse transcriptase–polymerase chain reaction (qRT‐PCR). Cell migration was compared in vitro and after grafting into nude – mdx mouse muscles. C2M9 clones produced high MMP‐9 and low MMP‐2, and migrated better than C2F clones, which secreted low MMP‐9, but overexpressed MMP‐2 and TIMP‐1. Improvement of C2F invasion by MMP‐9 and inhibition of C2M9 migration by MMP‐9 inhibitor I confirmed the role of MMP‐9 and pointed to potential inhibition by TIMP‐1. Higher complementation achieved by C2M9 grafts corroborated the beneficial effect of MMP‐9 overexpression. Modulation of MMP‐9 expression opens perspectives for improved efficacy of cell therapy for muscular dystrophies. Muscle Nerve, 2010
ABSTRACT Congenital ptosis with poor levator function is now managed by frontalis suspension techniques. While this procedure is better than those used in the past, serious shortcomings exist. A technique producing more normal lid function would be a beneficial addition to surgical management. Since congenital ptosis is thought to be a focal myopathy, we investigated the potential of myoblast transfer therapy in myopathic levator palpebrae superioris. Satellite cells harvested from temporalis muscle were grown as clones, labeled with Dil, and transplanted into experimentally myopathic levator muscle of the same animal. Within 2 weeks, the injected cells were found to be incorporated into muscle fibers within the levator basal lamina. The control side appeared myopathic with very little muscle regeneration. The presence of Dil labeled muscle fibers in the experimental muscles strongly suggests their origin from the injected cells. Electron microscopy of nearby sections showed these fibers to be maturing striated muscle. We feel that the development of this technique may make autogenous myoblast transfer therapy a useful treatment for congenital ptosis and other focal myopathies.
Infection of dendritic cells (DC) by human immunodeficiency virus (HIV) has been disputed. Employing a fluorescence-activated cell sorter, DC, identified by the absence of membrane markers for T, B, natural killer (NK) and monocytic cells and by high levels of MHC class II DR antigen, were shown to express low levels of CD4. Immunomagnetic beads were used to separate blood low density cells, which are enriched for DC, into CD4-positive and -negative populations. Examination of these cells by electron microscopy showed an increase in the percentage of cells with DC morphology in the CD4-positive fraction and a reduction in the CD4-negative fraction. Electron microscopy of semi-purified DC preparations infected in vitro for 5 days with HIV-1 revealed morphologically distinct veiled DC with mature virions on the cell surface and virus budding through the cell membrane. Further evidence for the growth of HIV in DC was provided by experiments in which DC were extensively depleted of contaminating lymphocytes and monocytes prior to infection. Estimation of provirus load by a nested PCR indicated that after 5 days an infection level of one provirus copy per five cells could be achieved. After 7 days the provirus copy number could exceed the cellular genome copy number, suggesting that some cells had more than one provirus. Infectious virus could not be demonstrated in these cultures after 24 h but was detected after 5 or 7 days. Infection of DC in the presence of antibodies against CD4 was inhibited and suggests infection occurs via a CD4-dependent pathway. These results confirm that DC are susceptible to HIV infection in vitro. The immunological consequences of DC infection in vivo may be significant in the pathogenesis of AIDS.
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.
Myoblasts transplanted into muscles of recipient mice mostly die, only a minor stem cell-like subpopulation surviving and participating in muscle regeneration. To investigate this phenomenon further, we used a retrovirus expressing beta-galactosidase to provide a unique marker for satellite-cell-derived muscle precursor cells, before transplanting them into myopathic mdx nu/nu mouse muscle. We employed inverse polymerase chain reaction to identify viral integrations, to follow the fate of clones present within the injected cells. Mass-infected cultures contained many marked clones, some of which contributed disproportionately to muscle regeneration. Although no particular clones showed overall predominance, some were present in more than one injected muscle, an eventuality unlikely to arise by chance. Conversely, in grafts of muscle precursor cells that had either been labelled as sparse satellite-cell derived cultures, or had been cloned, all clones were shown to be able to survive and form muscle in vivo. Moreover, all clones contributed to further generations of new-formed muscle fibres following a series of injuries administered to injected muscles, demonstrating that some cells of each clone had been retained as stem-cell-like muscle precursors. Furthermore, retrovirally marked satellite-cell-derived clones were derived from muscles that had been injected with marked muscle precursor cells. These cells formed muscle following their transplantation into a new host mouse, confirming their stem cell properties.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
