Heat shock treatment increases engraftment of transplanted human myoblasts into immunodeficient mice.

Myoblast transfer therapy (MTT) is a strategy that has been proposed to treat some striated muscle pathologies. However, the first therapeutic trials using this technique were unsuccessful due to the limited migration and early cell death of the injected myoblasts. Various strategies have been considered to increase myoblast survival in the host muscle after MTT. Overexpression of heat shock proteins (HSPs) in mouse myoblasts has been shown to improve cell resistance against apoptosis in vitro and in vivo. Our objective was to determine whether heat shock (HS) treatment increased the survival of human myoblasts leading to better participation of the injected cells in muscle regeneration. For this study, HS-treated human myoblasts were injected into the tibialis anterior (TA) muscles of immunodeficient RAG-/- gammaC-/- mice. TA muscles were excised at 24 hour and at 1 month after injection. Our results showed that HS treatment increased the expression of the hsp70 protein and protected the cells from apoptosis in vitro. HS treatment dramatically increased the number of human fibers present at 1 month after injection when compared with nontreated cells. Interestingly, HS treatment decreased apoptosis at 24 hour after human myoblast injection, but no differences were observed concerning proliferation, suggesting that the increased fiber formation among the HS-treated group was probably due to decreased cell death. These data suggested that HS treatment might be used in the clinical context to improve the success of MTT.

Myogenic stem cells: regeneration and cell therapy in human skeletal muscle.

Human skeletal muscle has been considered as an ideal target for cell-mediated therapy. However, the positive results obtained in dystrophic animal models using the resident precursor satellite cell population have been followed by discouraging evidences obtained in the clinical trials involving Duchenne muscular dystrophy patients. This text reviews the recent advances that many groups have achieved to identify from the stem cell compartment putative candidates for cell therapy. We focused our attention on stem cells with myogenic potential which might be able to improve transplantation efficiency and therefore could be used as a therapeutic tool for neuromuscular diseases.

The mitotic clock in skeletal muscle regeneration, disease and cell mediated gene therapy.

The regenerative capacity of skeletal muscle will depend on the number of available satellite cells and their proliferative capacity. We have measured both parameters in ageing, and have shown that although the proliferative capacity of satellite cells is decreasing during muscle growth, it then stabilizes in the adult, whereas the number of satellite cells decreases during ageing. We have also developed a model to evaluate the regenerative capacity of human satellite cells by implantation into regenerating muscles of immunodeficient mice. Using telomere measurements, we have shown that the proliferative capacity of satellite cells is dramatically decreased in muscle dystrophies, thus hampering the possibilities of autologous cell therapy. Immortalization by telomerase was unsuccessful, and we currently investigate the factors involved in cell cycle exits in human myoblasts. We have also observed that insulin-like growth factor-1 (IGF-1), a factor known to provoke hypertrophy, does not increase the proliferative potential of satellite cells, which suggests that hypertrophy is provoked by increasing the number of satellite cells engaged in differentiation, thus possibly decreasing the compartment of reserve cells. We conclude that autologous cell therapy can be applied to specific targets when there is a source of satellite cells which is not yet exhausted. This is the case of Oculo-Pharyngeal Muscular Dystrophy (OPMD), a late onset muscular dystrophy, and we participate to a clinical trial using autologous satellite cells isolated from muscles spared by the disease.

Myoblast transfer therapy: is there any light at the end of the tunnel?

Myoblast transfer therapy (MTT) was proposed in the 70's as a potential treatment for muscular dystrophies, based upon the early results obtained in mdx mice: dystrophin expression was restored in this model by intramuscular injections of normal myoblasts. These results were quickly followed by clinical trials for patients suffering from Duchenne Muscular Dystrophy (DMD) in the early 90's, based mainly upon intramuscular injections of allogenic myoblasts. The clinical benefits obtained from these trials were minimal, if any, and research programs concentrated then on the various pitfalls that hampered these clinical trials, leading to numerous failures. Several causes for these failures were identified in mouse models, including a massive cell death of myoblasts following their injection, adverse events involving the immune system and requiring immunosuppression and the adverse events linked to it, as well as a poor dispersion of the injected cells following their injection. It should be noted that these studies were conducted in mouse models, not taking into account the fundamental differences between mice and men. One of these differences concerns the regulation of proliferation, which is strictly limited by proliferative senescence in humans. Although this list is certainly not exhaustive, new therapeutic venues were then explored, such as the use of stem cells with myogenic potential, which have been described in various populations, including bone marrow, circulating blood or muscle itself. These stem cells presented the main advantage to be available and not exhausted by the numerous cycles of degeneration/regeneration which characterize muscle dystrophies. However, the different stem candidates have shown their limits in terms of efficiency to participate to the regeneration of the host. Another issue was raised by clinical trials involving the injection of autologous myoblasts in infacted hearts, which showed that limited targets could be aimed with autologous myoblasts, as long as enough spared muscle was available. This resulted in a clinical trial for the pharyngeal muscles of patients suffering from Oculo-Pharyngeal Muscular Dystrophy (OPMD). The results of this trial will not be available before 2 years, and a similar procedure is being studied for Fascio-Scapulo-Humeral muscular Dystrophy (FSHD). Concerning muscular dystrophies which leave very few muscles spared, such as DMD, other solutions must be found, which could include exon-skipping for the eligible patients, or even cell therapy using stem cells if some cell candidates with enough efficiency can be found. Recent results concerning mesoangioblasts or circulating AC133+ cells raise some reasonable hope, but still need further confirmations, since we have learned from the past to be cautious concerning a transfer of results from mice to humans.

Activity of 3-ketosphinganine synthase during differentiation and aging of neuronal cells in culture.

Changes in the enzyme 3-ketosphinganine synthase activity in rat cerebellar granule cells in culture were studied during differentiation and aging. The enzyme activity with palmitoyl-CoA and stearoyl-CoA, precursors of, respectively, C18-sphinganine and C20-sphinganine, was studied on the total cell homogenate using radioactive serine. The enzyme assay was performed by thin-layer chromatography (TLC) separation of the enzyme reaction mixture, and the resultant radioactive 3-ketosphinganine was identified by chromatographic comparison with a chemically synthesized 3-ketosphinganine, and quantified by determination of the TLC radioactivity distribution on the basis of the radioactivity content of cell lipid extract that was determined by scintillation counting. Using palmitoyl-CoA, the enzyme activity progressively increased from 40 to 54 pmol of 3-ketosphinganine/mg cell DNA per min in the first 8 days and then progressively decreased, and was 39 pmol of C18-(3-ketosphinganine)/mg cell DNA per min at day 22 in culture. For stearoyl-CoA the enzyme activity was very low at day one and then increased to a constant value of about 15 pmol of C20-(3-ketosphinganine)/mg cell DNA per min. These results are in good agreement with the finding that the ganglioside species that contain C18-sphingosine increase during cell differentiation and remain constant during cell aging, while the ganglioside species that contain C20-sphingosine continuously increase during both cell differentiation and aging.

Evaluation of the efficiency of an assay procedure for gangliosides in human serum.

An efficiency assessment of a ganglioside assay procedure was carried out on human serum gangliosides from healthy subjects of different sex and age. The analysis of the gangliosides, extracted with chloroform/methanol and purified by lipid partitioning, ion exchange column chromatographic separation and desalting procedures as described by Senn et al. (1989) Eur J Biochem 181: 657-62, was performed by HPTLC followed by densitometric quantification. The yield of the procedure, expressed as radioactivity recovery, was determined by adding GM3 ganglioside, tritium labelled at the sialic acid acetyl group and at the C3 position of sphingosine, to the lyophilized serum or by associating it with the serum lipoproteins. In spite of the fact that the extraction and purification procedures were performed exactly as described we found the radioactivity recovery to be variable (25-50%) and much lower than that proposed. Much of the radioactivity was found in the organic phase after lipid partitioning, whilst all the ganglioside purification steps led to some further loss. After the introduction of some modifications to the procedure the recovery improved, reaching 67-79%. The analyses on 33 samples of 5 ml showed a human serum ganglioside content of about 10 nmol ml-1 (as corrected for the recovery), and confirmed that GM3 ganglioside is the main component of the total serum ganglioside mixture.