An ex vivo gene therapy approach to treat muscular dystrophy using inducible pluripotent stem cells.

Duchenne muscular dystrophy is a progressive and incurable neuromuscular disease caused by genetic and biochemical defects of the dystrophin-glycoprotein complex. Here we show the regenerative potential of myogenic progenitors derived from corrected dystrophic induced pluripotent stem cells generated from fibroblasts of mice lacking both dystrophin and utrophin. We correct the phenotype of dystrophic induced pluripotent stem cells using a Sleeping Beauty transposon system carrying the micro-utrophin gene, differentiate these cells into skeletal muscle progenitors and transplant them back into dystrophic mice. Engrafted muscles displayed large numbers of micro-utrophin-positive myofibers, with biochemically restored dystrophin-glycoprotein complex and improved contractile strength. The transplanted cells seed the satellite cell compartment, responded properly to injury and exhibit neuromuscular synapses. We also detect muscle engraftment after systemic delivery of these corrected progenitors. These results represent an important advance towards the future treatment of muscular dystrophies using genetically corrected autologous induced pluripotent stem cells

Dominant lethal pathologies in male mice engineered to contain an X-linked DUX4 transgene

Facioscapulohumeral muscular dystrophy (FSHD) is an enigmatic disease associated with epigenetic alterations in the subtelomeric heterochromatin of the D4Z4 macrosatellite repeat. Each repeat unit encodes DUX4, a gene that is normally silent in most tissues. Besides muscular loss, most patients suffer retinal vascular telangiectasias. To generate an animal model, we introduced a doxycycline-inducible transgene encoding DUX4 and 3' genomic DNA into a euchromatic region of the mouse X chromosome. Without induction, DUX4 RNA was expressed at low levels in many tissues and animals displayed a variety of unexpected dominant leaky phenotypes, including male-specific lethality. Remarkably, rare live-born males expressed DUX4 RNA in the retina and presented a retinal vascular telangiectasia. By using doxycycline to induce DUX4 expression in satellite cells, we observed impaired myogenesis in vitro and in vivo. This mouse model, which shows pathologies due to FSHD-related D4Z4 sequences, is likely to be useful for testing anti-DUX4 therapies in FSHD

DUX4c, an FSHD candidate gene, interferes with myogenic regulators and abolishes myoblast differentiation

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disease. It maps to the D4Z4 repeat array at 4q35, and correlates with a repeat contraction which derepresses transcription of local genes. Which, if any, of these genes is pathogenic to muscle, and through what molecular mechanism is unknown. The present study investigates the function of one candidate gene, DUX4c, encoded by a truncated inverted D4Z4 element located 42 kb proximal to the D4Z4 repeats. Using a gain of function approach we tested DUX4c for toxicity and effects on differentiation in C2C12 myoblasts. DUX4c-expressing myoblasts appear morphologically normal but have reduced expression of myogenic regulators, including MyoD and Myf5. Consistent with this, DUX4c-expressing myoblasts are unable to differentiate into myotubes. Furthermore, overexpression of Myf5 or MyoD rescued myoblast differentiation, suggesting that reductions in expression of these regulators are the relevant DUX4c-induced physiological changes that inhibit differentiation. Our results suggest that upregulation of DUX4c can have a deleterious effect on muscle homeostasis and regeneration, and point to a possible role for DUX4c in the pathology of FSHD

Myogenic Cell Transplantation in Genetic and Acquired Diseases of Skeletal Muscle

From Frontiers via Jisc Publications RouterHistory: collection 2021, received 2021-04-29, accepted 2021-06-16, epub 2021-08-02Publication status: PublishedThis article will review myogenic cell transplantation for congenital and acquired diseases of skeletal muscle. There are already a number of excellent reviews on this topic, but they are mostly focused on a specific disease, muscular dystrophies and in particular Duchenne Muscular Dystrophy. There are also recent reviews on cell transplantation for inflammatory myopathies, volumetric muscle loss (VML) (this usually with biomaterials), sarcopenia and sphincter incontinence, mainly urinary but also fecal. We believe it would be useful at this stage, to compare the same strategy as adopted in all these different diseases, in order to outline similarities and differences in cell source, pre-clinical models, administration route, and outcome measures. This in turn may help to understand which common or disease-specific problems have so far limited clinical success of cell transplantation in this area, especially when compared to other fields, such as epithelial cell transplantation. We also hope that this may be useful to people outside the field to get a comprehensive view in a single review. As for any cell transplantation procedure, the choice between autologous and heterologous cells is dictated by a number of criteria, such as cell availability, possibility of in vitro expansion to reach the number required, need for genetic correction for many but not necessarily all muscular dystrophies, and immune reaction, mainly to a heterologous, even if HLA-matched cells and, to a minor extent, to the therapeutic gene product, a possible antigen for the patient. Finally, induced pluripotent stem cell derivatives, that have entered clinical experimentation for other diseases, may in the future offer a bank of immune-privileged cells, available for all patients and after a genetic correction for muscular dystrophies and other myopathies

Functional Myogenic Engraftment from Mouse iPS Cells

Direct reprogramming of adult fibroblasts to a pluripotent state has opened new possibilities for the generation of patient- and disease-specific stem cells. However the ability of induced pluripotent stem (iPS) cells to generate tissue that mediates functional repair has been demonstrated in very few animal models of disease to date. Here we present the proof of principle that iPS cells may be used effectively for the treatment of muscle disorders. We combine the generation of iPS cells with conditional expression of Pax7, a robust approach to derive myogenic progenitors. Transplantation of Pax7-induced iPS-derived myogenic progenitors into dystrophic mice results in extensive engraftment, which is accompanied by improved contractility of treated muscles. These findings demonstrate the myogenic regenerative potential of iPS cells and provide rationale for their future therapeutic application for muscular dystrophies

Funçao celular em individuos com exposição ocupacional ao mercurio

Orientador : Mary Luci de Souza QueirozDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: Neste trabalho investigamos alguns efeitos do mercúrio sobre o sistema imunológico. Foram estudados 51 trabalhadores expostos ao metal, assim como 51 indivíduos não-expostos (grupo controle). Os níveis urinários de mercúrio, determinados pelo método de absorção atômica, foram utilizados como índices biológicos de exposição. Foram estudadas a capacidade fagocitária e lítica de neutrófilos frente à Candida albicans e Candida pseudotropicalis, atividade quimiotática de neutrófilos e a capacidade proliferativa de linfócitos em resposta ao mitógeno fitohemaglutinina (PHA). O estudo da função esplênica foi efetuado através da contagem de hemácias com irregularidades de superfície (pits). Foi realizada uma 2ª avaliação destes parâmetros (intervalo de 6 meses). nos trabalhadores remanescentes, após uma melhoria efetuada nas condições de higiene da firma. Inicialmente, noventa e dois porcento dos trabalhadores estudados apresentavam níveis urinários de mercúrio abaixo dos limites de tolerância biológica (até 50 ug/g de creatinina). Após o intervalo de 6 meses, observamos, nos mesmos trabalhadores, uma redução significativa nestes níveis (p<0,05). Os nossos resultados na 1ª avaliação demonstraram que não houve alteração na capacidade fagocitária de neutrófilos frente aos 2 antígenos. Por outro lado, a atividade lítica apresentou-se reduzida em relação às duas espécies de Candida. Na 2ª avaliação, a atividade litica frente ao antígeno C.pseudotropicalis não. apresentou redução significativa em relação ao 1ª estudo. Por outro lado, em relação ao antígeno C.albicans, estas atividade apresentou-se ainda mais reduzida nesta 2ª avaliação, sendo essa tendência reforçada pela correlação linear observada entre tempo de exposição e atividade litica frente a este segundo antígeno. A atividade fagocitária manteve-se normal em relação aos dois antígenos. Foi também observada uma redução significativa na atividade quimiotática de neutrófilos, a qual manteve-se reduzida na 2ª avaliação. Não houve alteração significativa na capacidade proliferativa de linfócitos em resposta à PHA em presença de soro AB normal. No entanto, quando os linfócitos foram incubados em presença de soro autólogo, observamos uma redução na capacidade linfoproliferativa. A contagem de hemácias com irregularidades de superfície apresentou-se normal nestes trabalhadores (abaixo de 2%). Estes resultados indicam que níveis urinários de mercúrio considerados seguros no campo profissional não previnem o trabalhador exposto de efeitos tóxicos no sistema imunológiceAbstract: In this work some effects of mercury on the immune response were investigated. Fifty one male workers of a mercury producing plant were studied, as well as fifty one non-exposed controls. To monitor exposure we used urinary mercury levels (HgU), determined by the atomic absorption method. Phagocytosis and killing of Candida albicans and Candida pseudotropicalis by neutrophils, chemotaxis of neutrophils, lYmphocyte proliferation in the presence of the mitogen phytohemagglutinin(PHA) and the quantitation of red blood cell surface "pits" were measured.After improvement in the hygiene conditions in the factory, a new evaluation was performed, six months later, in the same workers. In the first evaluation, the results of mercury urinary concentrations .showed that 92% of the workers studied was below the threshold level value of 50ug/g of creatinine. After six months, these results were still lower (p<O.05) The results obtained in the first evaluation demonstrated that the phagocytic function of neutrophils in mercury-exposed workers did not differ from that of the controls. On the other hand, lysis of C. albicans and C.pseudotropicalis by neutrophils was significantly decreased. After 6 months, a greater impairment in the ability of neutrophils to kill Cpndi~a albicans was observed. The killing of Candida pseudotropicalis presented no changes, as compared to the previous evaluation . We also observed in the exposed group a significant impairment in the chemotaxis of neutrophils. This activity presented no changes, as compared to the previous evaluation. Blast lymphocyte transformation had no significant alterations, as compared to controls, when lymphocytes were incubated with AB serum. However, incubation of lymphocytes with autologous serum resulted in supression of the proliferation. The counting of pitted erythrocytes was not altered in these workers (below 2%). The relatively low level mercury absorption effects manifested by immunossupression indicates that immune dysfunction is a sensitive indicator of mercury exposureMestradoMestre em Farmacologi