RESUMO
BACKGROUND: Stem cell transplantation represents a potential therapeutic option for muscular dystrophies (MD). However, to date, most reports have utilized mouse models for recessive types of MD. Here we performed studies to determine whether myotonic dystrophy 1 (DM1), an autosomal dominant type of MD, could benefit from cell transplantation. METHODS: We injected human pluripotent stem (PS) cell-derived myogenic progenitors into the muscles of a novel mouse model combining immunodeficiency and skeletal muscle pathology of DM1 and investigated transplanted mice for engraftment as well as for the presence of RNA foci and alternative splicing pattern. FINDINGS: Engraftment was clearly observed in recipient mice, but unexpectedly, we detected RNA foci in donor-derived engrafted myonuclei. These foci proved to be pathogenic as we observed MBNL1 sequestration and abnormal alternative splicing in donor-derived transcripts. INTERPRETATION: It has been assumed that toxic CUG repeat-containing RNA forms foci in situ in the nucleus in which it is expressed, but these data suggest that CUG repeat-containing RNA may also exit the nucleus and traffic to other nuclei in the syncytial myofiber, where it can exert pathological effects. FUND: This project was supported by funds from the LaBonte/Shawn family and NIH grants R01 AR055299 and AR071439 (R.C.R.P.). R.M-G. was funded by CONACyT-Mexico (#394378).
Assuntos
Núcleo Celular/genética , Músculo Esquelético/metabolismo , Distrofia Miotônica/genética , RNA/genética , Processamento Alternativo , Animais , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Hospedeiro Imunocomprometido , Camundongos , Células Musculares/citologia , Células Musculares/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , RNA/administração & dosagemRESUMO
NEW FINDINGS: What is the central question of this study? This study investigated the role of the endogenous anti-inflammatory cytokine interleukin-10 in intense acute swimming-induced muscle mechanical hyperalgesia in mice. What is the main finding and its importance? Endogenous interleukin-10 has a key role in limiting exercise-induced muscle pain in a model presenting similarities to delayed-onset muscle soreness in mice. Interleukin-10 reduced muscle pain by diminishing leucocyte recruitment, hyperalgesic cytokine production, oxidative stress and myocyte damage. Interleukin-10 (IL-10) is an antihyperalgesic cytokine. In this study, IL-10-deficient (IL-10(-/-) ) mice were used to investigate the role of endogenous IL-10 in intense acute swimming-induced muscle mechanical hyperalgesia, which presents similarities with delayed-onset muscle soreness. An intense acute swimming session of 1 or 2 h induced significant muscle mechanical hyperalgesia in a time-dependent manner in wild-type mice compared with the sham group 24 h after the session, which was further increased in IL-10(-/-) mice (P Ë 0.05). Intraperitoneal treatment of wild-type mice with IL-10 (1-10 ng) reduced muscle mechanical hyperalgesia in a dose-dependent manner and reversed the enhanced muscle hyperalgesia in IL-10(-/-) mice (P Ë 0.05). The 2 h swimming session induced increases in tumour necrosis factor-α, interleukin-1ß and IL-10 production in the soleus muscle. However, tumour necrosis factor-α and interleukin-1ß production in the soleus muscle were even higher in IL-10(-/-) mice between 2 and 6 h after the stimulus (P Ë 0.05). There was no statistical difference in the levels of the antihyperalgesic cytokines interleukin-4, interleukin-5, interleukin-13 and transforming growth factor-ß between wild-type and IL-10(-/-) mice (P Ë 0.05). Interleukin-10 deficiency also resulted in increased myeloperoxidase activity, greater depletion of reduced glutathione levels, increased superoxide anion production and the maintenance of high plasma concentrations of creatine kinase (until 24 h after the swimming session) in soleus muscle (P Ë 0.05). These results demonstrate that endogenous IL-10 controls intense acute swimming-induced muscle mechanical hyperalgesia by limiting oxidative stress and cytokine production.
Assuntos
Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Interleucina-10/metabolismo , Músculo Esquelético/fisiopatologia , Mialgia/induzido quimicamente , Natação , Animais , Inflamação/metabolismo , Interleucina-10/genética , Masculino , Camundongos Endogâmicos C57BL , Células Musculares/citologia , Estresse Oxidativo/fisiologiaRESUMO
The combination of cell therapy with growth factors could be a useful approach to treat progressive muscular dystrophies. Here, we demonstrate, for the first time, that IGF-1 considerably enhances the myogenesis of human umbilical cord (UC) mesenchymal stromal cells (MSCs) in vitro and that IGF-1 enhances interaction and restoration of dystrophin expression in co-cultures of MSCs and muscle cells from Duchenne patients. In vivo studies showed that human MSCs were able to reach the skeletal muscle of LAMA2(dy/2j) dystrophic mice, through systemic delivery, without immunosuppression. Moreover, we showed, for the first time, that IGF-1 injected systemically together with MSCs markedly reduced muscle inflammation and fibrosis, and significantly improved muscle strength in dystrophic mice. Our results suggest that a combined treatment with IGF-1 and MSCs enhances efficiency of muscle repair and, therefore, should be further considered as a potential therapeutic approach in muscular dystrophies.
Assuntos
Fator de Crescimento Insulin-Like I/farmacologia , Laminina/metabolismo , Transplante de Células-Tronco Mesenquimais , Desenvolvimento Muscular/efeitos dos fármacos , Distrofia Muscular Animal/terapia , Animais , Diferenciação Celular/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Técnicas de Cocultura , Distrofina/biossíntese , Fibrose/terapia , Humanos , Inflamação/terapia , Laminina/genética , Células-Tronco Mesenquimais , Camundongos , Células Musculares/citologia , Células Musculares/metabolismo , Força Muscular/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Cordão Umbilical/citologiaRESUMO
We characterized the properties of the voltage-dependent K(+) currents I (to), I (Kr), and I (Ks) in isolated feline sino-atrial node (SAN) myocytes. I (to) activated rapidly and then inactivated with a single exponential and voltage-independent time course. Recovery from inactivation of I (to) followed a single exponential time course with τ = 21.1 ± 2.5 ms, at -80 mV. Steady-state inactivation relationship showed a V½ of inactivation at -47.9 ± 2.3 mV. These biophysical properties are similar to the fast I (to) phenotype of other mammals. I (Kr) exhibited typical negative slope conductance at test potentials > 0 mV and slow deactivation. I (Ks) activated very slowly. The functional contribution of I (to), I (Kr), and I (Ks) to the sustained pacemaking activity of feline SAN myocytes was analyzed. Similar to other mammals, I (to) underlies the initial repolarization phase of the SAN action potential, whereas I (Kr) and I (Ks) mediate repolarization back to the maximal diastolic potential. I (Kr) and I (Ks) also contribute to diastolic depolarization because of their slow deactivation kinetics. The I (Kr) specific blocker E-4031 and the I (Ks) blocker HMR 1556 significantly increased action potential duration, but had negligible effects on the maximum diastolic potential and only modest effects on the frequency of spontaneous activity, suggesting that each one of these two currents itself is capable of supporting action potential repolarization in the feline sinus node.
Assuntos
Células Musculares/metabolismo , Miocárdio/citologia , Canais de Potássio/fisiologia , Nó Sinoatrial/citologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Gatos , Células Cultivadas , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Técnicas de Patch-Clamp , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Nó Sinoatrial/fisiologiaRESUMO
OBJECTIVE: Pax3 and Pax7 are closely related genes that are involved in commitment of cells to a myogenic lineage during skeletal muscle development and regeneration. Several Pax3 and Pax7 transcripts are expressed from the genes, generating different isoforms with potentially distinct DNA binding and transactivation properties. The aim of this study was to investigate the implication of Pax3 and Pax7 C-terminal isoforms during myogenic differentiation and tumorigenesis, since fusions involving these genes are commonly associated with alveolar rhabdomyosarcoma (ARMS). METHODS: Uncommitted (mouse mesenchymal stem cells, MSCs) and committed (C2C12) myogenic precursor cells were stably transfected with PAX3/FKHR and PAXC7/ FKHR fusion genes. We analysed gene and protein expression comparing the newly generated cells with the parental cells, to determine the functional importance of Pax3 and Pax7 C-terminal isoforms. RESULTS: We found that the transcript Pax3c was expressed at low levels in undifferentiated C2C12 and MSCs cells, but its expression levels increased considerably at later stages of differentiation. However, expression levels of Pax3d transcript increased only slightly after differentiation. Pax7 transcripts, present before differentiation in committed C2C12 cells, but absent in uncommitted MSCs, increased noticeably in MSCs after differentiation. We also found that the presence of PAX/FKHR fusions prevented both C2C12 and MSC cells from terminal myogenic differentiation and increased the expression of discrete endogenous Pax3/7 transcripts, in particular Pax3d and Pax7B. CONCLUSIONS: Our results suggest that both Pax3 and Pax7 transcripts are required for commitment of cells to the myogenic lineage, with each transcript having a distinct role. More specifically, the Pax3c isoform may be required for terminal myogenic differentiation whereas the Pax3d isoform may be involved in undifferentiated cell maintenance and/or proliferation.
Assuntos
Diferenciação Celular/fisiologia , Células Musculares/citologia , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma Alveolar/genética , Animais , Linhagem Celular , Linhagem da Célula/fisiologia , Humanos , Imuno-Histoquímica , Células-Tronco Mesenquimais/citologia , Camundongos , Células Musculares/metabolismo , Fator de Transcrição PAX3 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rabdomiossarcoma Alveolar/metabolismo , TransfecçãoRESUMO
BACKGROUND: The possibility of using stem cells for regenerative medicine has opened a new field of investigation. The search for sources to obtain multipotent stem cells from discarded tissues or through non-invasive procedures is of great interest. It has been shown that mesenchymal stem cells (MSCs) obtained from umbilical cords, dental pulp and adipose tissue, which are all biological discards, are able to differentiate into muscle, fat, bone and cartilage cell lineages. The aim of this study was to isolate, expand, characterize and assess the differentiation potential of MSCs from human fallopian tubes (hFTs). METHODS: Lineages of hFTs were expanded, had their karyotype analyzed, were characterized by flow cytometry and underwent in vitro adipogenic, chondrogenic, osteogenic, and myogenic differentiation. RESULTS: Here we show for the first time that hFTs, which are discarded after some gynecological procedures, are a rich additional source of MSCs, which we designated as human tube MSCs (htMSCs). CONCLUSION: Human tube MSCs can be easily isolated, expanded in vitro, present a mesenchymal profile and are able to differentiate into muscle, fat, cartilage and bone in vitro.
Assuntos
Tubas Uterinas/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Adipócitos/citologia , Adulto , Diferenciação Celular/fisiologia , Linhagem da Célula , Proliferação de Células , Separação Celular , Células Cultivadas , Condrócitos/citologia , Tubas Uterinas/cirurgia , Feminino , Humanos , Cariotipagem , Células-Tronco Mesenquimais/fisiologia , Células Musculares/citologia , Osteoblastos/citologiaRESUMO
OBJECTIVES: In this study, we aimed at determining whether human immature dental pulp stem cells (hIDPSC) would be able to contribute to different cell types in mouse blastocysts without damaging them. Also, we analysed whether these blastocysts would progress further into embryogenesis when implanted to the uterus of foster mice, and develop human/mouse chimaera with retention of hIDPSC derivates and their differentiation. MATERIALS AND METHODS: hIDPSC and mouse blastocysts were used in this study. Fluorescence staining of hIDPSC and injection into mouse blastocysts, was performed. Histology, immunohistochemistry, fluorescence in situ hybridization and confocal microscopy were carried out. RESULTS AND CONCLUSION: hIDPSC showed biological compatibility with the mouse host environment and could survive, proliferate and contribute to the inner cell mass as well as to the trophoblast cell layer after introduction into early mouse embryos (n = 28), which achieved the hatching stage following 24 and 48 h in culture. When transferred to foster mice (n = 5), these blastocysts with hIDPSC (n = 57) yielded embryos (n = 3) and foetuses (n = 6); demonstrating presence of human cells in various organs, such as brain, liver, intestine and hearts, of the human/mouse chimaeras. We verified whether hIDPSC would also be able to differentiate into specific cell types in the mouse environment. Contribution of hIDPSC in at least two types of tissues (muscles and epithelial), was confirmed. We showed that hIDPSC survived, proliferated and differentiated in mouse developing blastocysts and were capable of producing human/mouse chimaeras.
Assuntos
Células-Tronco Adultas/citologia , Polpa Dentária/citologia , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/fisiologia , Feto/citologia , Quimeras de Transplante/embriologia , Células-Tronco Adultas/transplante , Estruturas Animais/citologia , Estruturas Animais/embriologia , Estruturas Animais/metabolismo , Animais , Blastocisto/citologia , Diferenciação Celular/fisiologia , Cromossomos Humanos Y/química , Transferência Embrionária , Embrião de Mamíferos/embriologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Feminino , Feto/embriologia , Feto/metabolismo , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos , Células Musculares/citologia , Células Musculares/metabolismo , Músculos/citologia , Músculos/embriologia , Músculos/metabolismo , Quimeras de Transplante/metabolismoRESUMO
BACKGROUND INFORMATION: DMD (Duchenne muscular dystrophy) is a devastating X-linked disorder characterized by progressive muscle degeneration and weakness. The use of cell therapy for the repair of defective muscle is being pursued as a possible treatment for DMD. Mesenchymal stem cells have the potential to differentiate and display a myogenic phenotype in vitro. Since liposuctioned human fat is available in large quantities, it may be an ideal source of stem cells for therapeutic applications. ASCs (adipose-derived stem cells) are able to restore dystrophin expression in the muscles of mdx (X-linked muscular dystrophy) mice. However, the outcome when these cells interact with human dystrophic muscle is still unknown. RESULTS: We show here that ASCs participate in myotube formation when cultured together with differentiating human DMD myoblasts, resulting in the restoration of dystrophin expression. Similarly, dystrophin was induced when ASCs were co-cultivated with DMD myotubes. Experiments with GFP (green fluorescent protein)-positive ASCs and DAPI (4',6-diamidino-2-phenylindole)-stained DMD myoblasts indicated that ASCs participate in human myogenesis through cellular fusion. CONCLUSIONS: These results show that ASCs have the potential to interact with dystrophic muscle cells, restoring dystrophin expression of DMD cells in vitro. The possibility of using adipose tissue as a source of stem cell therapies for muscular diseases is extremely exciting.
Assuntos
Tecido Adiposo/citologia , Distrofina/metabolismo , Células-Tronco Multipotentes/citologia , Células Musculares/citologia , Desenvolvimento Muscular , Distrofia Muscular de Duchenne/metabolismo , Células-Tronco/citologia , Western Blotting , Técnicas de Cultura de Células , Diferenciação Celular , Separação Celular , Técnicas de Cocultura , Citometria de Fluxo , Expressão Gênica , Humanos , Fibras Musculares Esqueléticas/citologia , Mioblastos/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução GenéticaRESUMO
Cholesterol is one of the major lipids of plasma membranes. Recently, we have shown that cholesterol depletion by methyl-beta-cyclodextrin (M beta CD) induces the activation of the Wnt/beta-catenin pathway and enhances myogenic differentiation. Here, we show that M beta CD-conditioned media accelerates myogenesis in a similar way as M beta CD does, suggesting that the effects induced by M beta CD could be caused by soluble factors present in the culture medium. Soluble Wnt-3 protein is significantly enhanced in M beta CD-conditioned medium. Wnt-3a-enriched media induces myogenesis as much as M beta CD does, whereas Wnt-5a-enriched media inhibits. We suggest that Wnt-3a is involved in the myogenic induction observed after cholesterol depletion.
Assuntos
Diferenciação Celular , Colesterol/deficiência , Células Musculares/citologia , Desenvolvimento Muscular , Proteínas Wnt/metabolismo , Animais , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Células Cultivadas , Embrião de Galinha , Meios de Cultivo Condicionados/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Peso Molecular , Células Musculares/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Ratos , Solubilidade/efeitos dos fármacos , Proteína Wnt3 , Proteína Wnt3A , beta-Ciclodextrinas/farmacologiaRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) patients carry contractions of the D4Z4-tandem repeat array on chromosome 4q35. Decrease in D4Z4 copy number is thought to alter a chromatin structure and activate expression of neighboring genes. D4Z4 contains a putative double-homeobox gene called DUX4. We identified DUX4 mRNAs in cells transfected with genomic fragments containing the DUX4 gene. Using RT-PCR we also recognized expressed DUX4 mRNAs in primary FSHD myoblasts. Polyclonal antibodies raised against specific DUX4 peptides detected the DUX4 protein in cells transfected with D4Z4 elements. DUX4 localizes in the nucleus of cells transfected with CMV-DUX4 expression vectors. A DUX4-related protein is endogenously expressed in nuclei of adult and fetal human rhabdomyosarcoma cell lines. Overexpression of DUX4 induces cell death, induces caspase 3/7 activity and alters emerin distribution at the nuclear envelope. We propose that DUX4-mediated cell death contributes to the pathogenic pathway in FSHD.