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1.
Proc Natl Acad Sci U S A ; 121(31): e2314760121, 2024 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-39052834

RESUMEN

Transceptors, solute transporters that facilitate intracellular entry of molecules and also initiate intracellular signaling events, have been primarily studied in lower-order species. Ammonia, a cytotoxic endogenous metabolite, is converted to urea in hepatocytes for urinary excretion in mammals. During hyperammonemia, when hepatic metabolism is impaired, nonureagenic ammonia disposal occurs primarily in skeletal muscle. Increased ammonia uptake in skeletal muscle is mediated by a membrane-bound, 12 transmembrane domain solute transporter, Rhesus blood group-associated B glycoprotein (RhBG). We show that in addition to its transport function, RhBG interacts with myeloid differentiation primary response-88 (MyD88) to initiate an intracellular signaling cascade that culminates in activation of NFκB. We also show that ammonia-induced MyD88 signaling is independent of the canonical toll-like receptor-initiated mechanism of MyD88-dependent NFκB activation. In silico, in vitro, and in situ experiments show that the conserved cytosolic J-domain of the RhBG protein interacts with the Toll-interleukin-1 receptor (TIR) domain of MyD88. In skeletal muscle from human patients, human-induced pluripotent stem cell-derived myotubes, and myobundles show an interaction of RhBG-MyD88 during hyperammonemia. Using complementary experimental and multiomics analyses in murine myotubes and mice with muscle-specific RhBG or MyD88 deletion, we show that the RhBG-MyD88 interaction is essential for the activation of NFkB but not ammonia transport. Our studies show a paradigm of substrate-dependent regulation of transceptor function with the potential for modulation of cellular responses in mammalian systems by decoupling transport and signaling functions of transceptors.


Asunto(s)
Amoníaco , Proteínas de Transporte de Membrana , Factor 88 de Diferenciación Mieloide , FN-kappa B , Transducción de Señal , Animales , Humanos , Ratones , Amoníaco/metabolismo , Hiperamonemia/metabolismo , Hiperamonemia/genética , Ratones Noqueados , Músculo Esquelético/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Factor 88 de Diferenciación Mieloide/genética , FN-kappa B/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo
2.
PLoS Biol ; 22(3): e3002503, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38478490

RESUMEN

Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices, and microfluidic systems to larger constructs such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques, equipment and tools required for their manufacture, and the expertise necessary, biological and biomedical labs tend more often to rely on already made devices. Indeed, commercially developed devices are available for a variety of applications but are often costly and, importantly, lack the potential for customisation by each individual lab. The last point is quite crucial, as often experiments in wet labs are adapted to whichever design is already available rather than designing and fabricating custom systems that perfectly fit the biological question. This combination of factors still restricts widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent advances in bioengineering and microfabrication aimed at solving these issues, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised a low-cost and highly reproducible microfabrication pipeline. This is thought specifically for biomedical and biological wet labs with not prior experience in the field, which will enable them to generate a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with limited expertise in those techniques and enables the manufacture of complex devices across the µm to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D-printed constructs for PDMS curing, using a combination of polymerisation steps, washes, and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilisation of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multipart hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.


Asunto(s)
Técnicas de Cultivo de Célula , Microtecnología , Microfluídica/métodos , Impresión Tridimensional , Dispositivos Laboratorio en un Chip
3.
Stem Cell Res ; 69: 103079, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36989620

RESUMEN

Centronuclear myopathies (CNMs) are a group of inherited rare muscle disorders characterised by the abnormal position of the nucleus in the center of the muscle fiber. One of CNM is the X-Linked Myotubular Myopathy, caused by mutations in the myotubularin (MTM1) gene (XLMTM), characterised by profound muscle hypotonia and weakness, severe bulbar and respiratory involvement. Here, we generated an induced pluripotent stem cell (iPSC) line from a patient with a severe form of XLMTM. Dermal fibroblasts were reprogrammed to pluripotency using a non-integrating mRNA-based protocol. This new MTM1-mutant iPSC line could facilitate disease-modelling and therapy development studies for XLMTM.


Asunto(s)
Células Madre Pluripotentes Inducidas , Miopatías Estructurales Congénitas , Humanos , Fibras Musculares Esqueléticas , Mutación/genética , Miopatías Estructurales Congénitas/genética , Núcleo Celular , Músculo Esquelético
4.
Nat Protoc ; 18(4): 1337-1376, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36792780

RESUMEN

Skeletal muscle is a complex tissue composed of multinucleated myofibers responsible for force generation that are supported by multiple cell types. Many severe and lethal disorders affect skeletal muscle; therefore, engineering models to reproduce such cellular complexity and function are instrumental for investigating muscle pathophysiology and developing therapies. Here, we detail the modular 3D bioengineering of multilineage skeletal muscles from human induced pluripotent stem cells, which are first differentiated into myogenic, neural and vascular progenitor cells and then combined within 3D hydrogels under tension to generate an aligned myofiber scaffold containing vascular networks and motor neurons. 3D bioengineered muscles recapitulate morphological and functional features of human skeletal muscle, including establishment of a pool of cells expressing muscle stem cell markers. Importantly, bioengineered muscles provide a high-fidelity platform to study muscle pathology, such as emergence of dysmorphic nuclei in muscular dystrophies caused by mutant lamins. The protocol is easy to follow for operators with cell culture experience and takes between 9 and 30 d, depending on the number of cell lineages in the construct. We also provide examples of applications of this advanced platform for testing gene and cell therapies in vitro, as well as for in vivo studies, providing proof of principle of its potential as a tool to develop next-generation neuromuscular or musculoskeletal therapies.


Asunto(s)
Células Madre Pluripotentes Inducidas , Células Satélite del Músculo Esquelético , Humanos , Músculo Esquelético/metabolismo , Diferenciación Celular/fisiología , Linaje de la Célula
5.
EMBO Mol Med ; 14(10): e14526, 2022 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-36161772

RESUMEN

Muscle satellite stem cells (MuSCs) are responsible for skeletal muscle growth and regeneration. Despite their differentiation potential, human MuSCs have limited in vitro expansion and in vivo migration capacity, limiting their use in cell therapies for diseases affecting multiple skeletal muscles. Several protocols have been developed to derive MuSC-like progenitors from human induced pluripotent stem (iPS) cells (hiPSCs) to establish a source of myogenic cells with controllable proliferation and differentiation. However, current hiPSC myogenic derivatives also suffer from limitations of cell migration, ultimately delaying their clinical translation. Here we use a multi-disciplinary approach including bioinformatics and tissue engineering to show that DLL4 and PDGF-BB improve migration of hiPSC-derived myogenic progenitors. Transcriptomic analyses demonstrate that this property is conserved across species and multiple hiPSC lines, consistent with results from single cell motility profiling. Treated cells showed enhanced trans-endothelial migration in transwell assays. Finally, increased motility was detected in a novel humanised assay to study cell migration using 3D artificial muscles, harnessing advanced tissue modelling to move hiPSCs closer to future muscle gene and cell therapies.


Asunto(s)
Células Madre Pluripotentes Inducidas , Becaplermina/metabolismo , Diferenciación Celular , Humanos , Desarrollo de Músculos , Músculo Esquelético/metabolismo , Mioblastos
6.
Exp Cell Res ; 416(2): 113133, 2022 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-35427601

RESUMEN

Engineering models of human skeletal muscle tissue provides unique translational opportunities to investigate and develop therapeutic strategies for acute muscle injuries, and to establish personalised and precision medicine platforms for in vitro studies of severe neuromuscular and musculoskeletal disorders. Several myogenic and non-myogenic cell types can be isolated, generated, amplified and combined with scaffolds and biomaterials to achieve this aim. Novel bio-fabrication strategies, which include exogenous stimuli to enhance tissue maturation, promise to achieve an ever-increasing degree of tissue functionalisation both in vivo and in vitro. Here we review recent advances, current challenges and future perspectives to build human skeletal muscle tissue "in a dish", focusing on the cellular constituents and on applications for in vitro disease modelling. We also briefly discuss the impact that emerging technologies such as 3D bioprinting, organ-on-chip and organoids might have to circumvent technical hurdles in future studies.


Asunto(s)
Bioimpresión , Ingeniería de Tejidos , Bioingeniería , Humanos , Músculo Esquelético , Andamios del Tejido
7.
J Cachexia Sarcopenia Muscle ; 13(2): 1360-1372, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35083887

RESUMEN

BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by DMD mutations leading to dystrophin loss. Full-length Dp427 is the primary dystrophin isoform expressed in muscle and is also expressed in the central nervous system (CNS). Two shorter isoforms, Dp140 and Dp71, are highly expressed in the CNS. While a role for Dp140 and Dp71 on DMD CNS comorbidities is well known, relationships between mutations expected to disrupt Dp140 and Dp71 and motor outcomes are not. METHODS: Functional outcome data from 387 DMD boys aged 4-15 years were subdivided by DMD mutation expected effects on dystrophin isoform expression; Group 1 (Dp427 absent, Dp140/Dp71 present, n = 201); Group 2 (Dp427/Dp140 absent, Dp71 present, n = 152); and Group 3 (Dp427/Dp140/Dp71 absent, n = 34). Relationships between isoform group and North Star ambulatory assessment (NSAA) scores, 10 m walk/run velocities and rise time velocities were explored using regression analysis. Western blot analysis was used to study Dp427, Dp140 and Dp71 production in myogenic cells (control and DMD human), control skeletal muscle, DMD skeletal muscle from the three isoform groups and cerebral cortex from mice (wild-type and DMD models). Grip strength and rotarod running test were studied in wild-type mice and DMD mouse models. DMD mouse models were mdx (Dp427 absent, Dp140/Dp71 present), mdx52 (Dp427/Dp140 absent, Dp71 present) and DMD-null (lacking all isoforms). RESULTS: In DMD boys, mean NSAA scores at 5 years of age were 6.1 points lower in Group 3 than Group 1 (P < 0.01) and 4.9 points lower in Group 3 than Group 2 (P = 0.05). Mean peak NSAA scores were 4.0 points lower in Group 3 than Group 1 (P < 0.01) and 1.6 points lower in Group 2 than Group 1 (P = 0.04). Mean four-limb grip strength was 1.5 g/g lower in mdx52 than mdx mice (P = 0.003) and 1.5 g/g lower in DMD-null than mdx mice (P = 0.002). Dp71 was produced in myogenic cells (control and DMD human) and skeletal muscle from humans in Groups 1 and 2 and mdx mice, but not skeletal muscle from human controls, myogenic cells and skeletal muscle from humans in Group 3 or skeletal muscle from wild-type, mdx52 or DMD-null mice. CONCLUSIONS: Our results highlight the importance of considering expected effects of DMD mutations on dystrophin isoform production when considering patterns of DMD motor impairment and the implications for clinical practice and clinical trials. Our results suggest a complex relationship between dystrophin isoforms expressed in the brain and DMD motor function.


Asunto(s)
Distrofina , Distrofia Muscular de Duchenne , Animales , Distrofina/genética , Distrofina/metabolismo , Humanos , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
8.
Front Genet ; 13: 1056114, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36685855

RESUMEN

In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had accumulated that during muscle development and regeneration things may be more complex than a simple sequence of binary choices (e.g., dorsal vs. ventral somite). LacZ expressing fibroblasts could fuse with unlabelled myoblasts but not among themselves or with other cell types. Bone marrow derived, circulating progenitors were able to participate in muscle regeneration, though in very small percentage. Searching for the embryonic origin of these progenitors, we identified them as originating at least in part from the embryonic aorta and, at later stages, from the microvasculature of skeletal muscle. While continuing to investigate origin and fate of MABs, the fact that they could be expanded in vitro (also from human muscle) and cross the vessel wall, suggested a protocol for the cell therapy of muscular dystrophies. We tested this protocol in mice and dogs before proceeding to the first clinical trial on Duchenne Muscular Dystrophy patients that showed safety but minimal efficacy. In the last years, we have worked to overcome the problem of low engraftment and tried to understand their role as auxiliary myogenic progenitors during development and regeneration.

9.
Glia ; 70(3): 466-490, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34773297

RESUMEN

In addition to progressive muscular degeneration due to dystrophin mutations, 1/3 of Duchenne muscular dystrophy (DMD) patients present cognitive deficits. However, there is currently an incomplete understanding about the function of the multiple dystrophin isoforms in human brains. Here, we tested the hypothesis that dystrophin deficiency affects glial function in DMD and could therefore contribute to neural impairment. We investigated human dystrophin isoform expression with development and differentiation and response to damage in human astrocytes from control and induced pluripotent stem cells from DMD patients. In control cells, short dystrophin isoforms were up-regulated with development and their expression levels changed differently upon neuronal and astrocytic differentiation, as well as in 2-dimensional versus 3-dimensional astrocyte cultures. All DMD-astrocytes tested displayed altered morphology, proliferative activity and AQP4 expression. Furthermore, they did not show any morphological change in response to inflammatory stimuli and their number was significantly lower as compared to stimulated healthy astrocytes. Finally, DMD-astrocytes appeared to be more sensitive than controls to oxidative damage as shown by their increased cell death. Behavioral and metabolic defects in DMD-astrocytes were consistent with gene pathway dysregulation shared by lines with different mutations as demonstrated by bulk RNA-seq analysis. Together, our DMD model provides evidence for altered astrocyte function in DMD suggesting that defective astrocyte responses may contribute to neural impairment and might provide additional potential therapeutic targets.


Asunto(s)
Células Madre Pluripotentes Inducidas , Distrofia Muscular de Duchenne , Astrocitos/metabolismo , Diferenciación Celular , Distrofina/genética , Distrofina/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo
10.
Cell Rep ; 36(8): 109601, 2021 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-34433058

RESUMEN

Cofilins are important for the regulation of the actin cytoskeleton, sarcomere organization, and force production. The role of cofilin-1, the non-muscle-specific isoform, in muscle function remains unclear. Mutations in LMNA encoding A-type lamins, intermediate filament proteins of the nuclear envelope, cause autosomal Emery-Dreifuss muscular dystrophy (EDMD). Here, we report increased cofilin-1 expression in LMNA mutant muscle cells caused by the inability of proteasome degradation, suggesting a protective role by ERK1/2. It is known that phosphorylated ERK1/2 directly binds to and catalyzes phosphorylation of the actin-depolymerizing factor cofilin-1 on Thr25. In vivo ectopic expression of cofilin-1, as well as its phosphorylated form on Thr25, impairs sarcomere structure and force generation. These findings present a mechanism that provides insight into the molecular pathogenesis of muscular dystrophies caused by LMNA mutations.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Cofilina 1/metabolismo , Destrina/metabolismo , Lamina Tipo A/metabolismo , Laminopatías/metabolismo , Músculo Estriado/metabolismo , Sarcómeros/metabolismo , Adolescente , Adulto , Animales , Línea Celular , Niño , Humanos , Lamina Tipo A/genética , Laminopatías/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Músculo Estriado/patología , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/metabolismo , Mutación , Fosforilación , Transducción de Señal , Adulto Joven
11.
Curr Opin Cell Biol ; 73: 92-104, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34384976

RESUMEN

Advanced in vitro models of human skeletal muscle tissue are increasingly needed to model complex developmental dynamics and disease mechanisms not recapitulated in animal models or in conventional monolayer cell cultures. There has been impressive progress towards creating such models by using tissue engineering approaches to recapitulate a range of physical and biochemical components of native human skeletal muscle tissue. In this review, we discuss recent studies focussed on developing complex in vitro models of human skeletal muscle beyond monolayer cell cultures, involving skeletal myogenic differentiation from human primary myoblasts or pluripotent stem cells, often in the presence of structural scaffolding support. We conclude with our outlook on the future of advanced skeletal muscle three-dimensional cultures (e.g. organoids and biofabrication) to produce physiologically and clinically relevant platforms for disease modelling and therapy development in musculoskeletal and neuromuscular disorders.


Asunto(s)
Desarrollo de Músculos , Organoides , Animales , Diferenciación Celular , Humanos , Músculo Esquelético , Ingeniería de Tejidos
12.
Mol Ther Nucleic Acids ; 23: 629-639, 2021 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-33552683

RESUMEN

Genetic engineering of induced pluripotent stem cells (iPSCs) holds great promise for gene and cell therapy as well as drug discovery. However, there are potential concerns regarding the safety and control of gene expression using conventional vectors such as viruses and plasmids. Although human artificial chromosome (HAC) vectors have several advantages as a gene delivery vector, including stable episomal maintenance and the ability to carry large gene inserts, the full potential of HAC transfer into iPSCs still needs to be explored. Here, we provide evidence of a HAC transfer into human iPSCs by microcell-mediated chromosome transfer via measles virus envelope proteins for various applications, including gene and cell therapy, establishment of versatile human iPSCs capable of gene loading and differentiation into T cells, and disease modeling for aneuploidy syndrome. Thus, engineering of human iPSCs via desired HAC vectors is expected to be widely applied in biomedical research.

13.
EMBO Mol Med ; 12(12): e12357, 2020 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-33210465

RESUMEN

Directional cell migration is a critical process underlying morphogenesis and post-natal tissue regeneration. During embryonic myogenesis, migration of skeletal myogenic progenitors is essential to generate the anlagen of limbs, diaphragm and tongue, whereas in post-natal skeletal muscles, migration of muscle satellite (stem) cells towards regions of injury is necessary for repair and regeneration of muscle fibres. Additionally, safe and efficient migration of transplanted cells is critical in cell therapies, both allogeneic and autologous. Although various myogenic cell types have been administered intramuscularly or intravascularly, functional restoration has not been achieved yet in patients with degenerative diseases affecting multiple large muscles. One of the key reasons for this negative outcome is the limited migration of donor cells, which hinders the overall cell engraftment potential. Here, we review mechanisms of myogenic stem/progenitor cell migration during skeletal muscle development and post-natal regeneration. Furthermore, strategies utilised to improve migratory capacity of myogenic cells are examined in order to identify potential treatments that may be applied to future transplantation protocols.


Asunto(s)
Movimiento Celular , Desarrollo de Músculos , Músculo Esquelético/citología , Animales , Humanos , Fibras Musculares Esqueléticas/citología , Células Satélite del Músculo Esquelético/citología
14.
Stem Cell Res ; 43: 101688, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32087527

RESUMEN

Duchenne muscular dystrophy (DMD) is the most common paediatric muscular dystrophy and is caused by mutations in the DYSTROPHIN gene. We generated two induced pluripotent stem cell (iPSC) lines from DMD patients with nonsense mutations in exons 68 (UCLi011-A) or 70 (UCLi012-A) by transfecting reprogramming mRNAs. Both mutations affect expression of all dystrophin isoforms. iPSCs expressed pluripotency-associated markers, differentiated into cells of the three germ layers in vitro and had normal karyotypes. The selected mutations are potentially amenable to read-through therapies, exon-skipping and gene-editing. These new iPSCs are also relevant to study DYSTROPHIN role in tissues other than skeletal muscle.


Asunto(s)
Distrofina/metabolismo , Exones/genética , Distrofia Muscular de Duchenne/genética , Isoformas de Proteínas/metabolismo , Animales , Diferenciación Celular , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Transfección
15.
J Cell Biol ; 218(9): 2826-2828, 2019 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-31427369

RESUMEN

Lamins A and C are intermediate filaments that provide structural support to the nuclear envelope and regulate gene expression. In this issue, Bertero et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201902117) report that although lamin A/C haploinsufficient cardiomyocytes show disease-associated phenotypes, those changes cannot be explained by alterations in chromatin compartmentalization.


Asunto(s)
Cromatina , Células Madre Pluripotentes Inducidas , Haploinsuficiencia , Lamina Tipo A/genética , Membrana Nuclear
16.
Adv Exp Med Biol ; 1147: 319-344, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31147885

RESUMEN

The muscular dystrophies are an heterogeneous group of inherited myopathies characterised by the progressive wasting of skeletal muscle tissue. Pericytes have been shown to make muscle in vitro and to contribute to skeletal muscle regeneration in several animal models, although recent data has shown this to be controversial. In fact, some pericyte subpopulations have been shown to contribute to fibrosis and adipose deposition in muscle. In this chapter, we explore the identity and the multifaceted role of pericytes in dystrophic muscle, potential therapeutic applications and the current need to overcome the hurdles of characterisation (both to identify pericyte subpopulations and track cell fate), to prevent deleterious differentiation towards myogenic-inhibiting subpopulations, and to improve cell proliferation and engraftment efficacy.


Asunto(s)
Distrofias Musculares , Pericitos , Animales , Diferenciación Celular , Desarrollo de Músculos , Músculo Esquelético , Regeneración
17.
Stem Cell Reports ; 12(3): 461-473, 2019 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-30745033

RESUMEN

Satellite cells are responsible for skeletal muscle regeneration. Upon activation, they proliferate as transient amplifying myoblasts, most of which fuse into regenerating myofibers. Despite their remarkable differentiation potential, these cells have limited migration capacity, which curtails clinical use for widespread forms of muscular dystrophy. Conversely, skeletal muscle perivascular cells have less myogenic potential but better migration capacity than satellite cells. Here we show that modulation of Notch and PDGF pathways, involved in developmental specification of pericytes, induces perivascular cell features in adult mouse and human satellite cell-derived myoblasts. DLL4 and PDGF-BB-treated cells express markers of perivascular cells and associate with endothelial networks while also upregulating markers of satellite cell self-renewal. Moreover, treated cells acquire trans-endothelial migration ability while remaining capable of engrafting skeletal muscle upon intramuscular transplantation. These results extend our understanding of muscle stem cell fate plasticity and provide a druggable pathway with clinical relevance for muscle cell therapy.


Asunto(s)
Biomarcadores/metabolismo , Movimiento Celular/fisiología , Receptores Notch/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Transducción de Señal/fisiología , Células Madre/metabolismo , Animales , Células Endoteliales/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Desarrollo de Músculos/fisiología , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Pericitos/metabolismo , Regeneración/fisiología , Regulación hacia Arriba/fisiología
18.
Front Physiol ; 9: 1332, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30405424

RESUMEN

Laminopathies are a clinically heterogeneous group of disorders caused by mutations in LMNA. The main proteins encoded by LMNA are Lamin A and C, which together with Lamin B1 and B2, form the nuclear lamina: a mesh-like structure located underneath the inner nuclear membrane. Laminopathies show striking tissue specificity, with subtypes affecting striated muscle, peripheral nerve, and adipose tissue, while others cause multisystem disease with accelerated aging. Although several pathogenic mechanisms have been proposed, the exact pathophysiology of laminopathies remains unclear, compounded by the rarity of these disorders and lack of easily accessible cell types to study. To overcome this limitation, we used induced pluripotent stem cells (iPSCs) from patients with skeletal muscle laminopathies such as LMNA-related congenital muscular dystrophy and limb-girdle muscular dystrophy 1B, to model disease phenotypes in vitro. iPSCs can be derived from readily accessible cell types, have unlimited proliferation potential and can be differentiated into cell types that would otherwise be difficult and invasive to obtain. iPSC lines from three skeletal muscle laminopathy patients were differentiated into inducible myogenic cells and myotubes. Disease-associated phenotypes were observed in these cells, including abnormal nuclear shape and mislocalization of nuclear lamina proteins. Nuclear abnormalities were less pronounced in monolayer cultures of terminally differentiated skeletal myotubes than in proliferating myogenic cells. Notably, skeletal myogenic differentiation of LMNA-mutant iPSCs in artificial muscle constructs improved detection of myonuclear abnormalities compared to conventional monolayer cultures across multiple pathogenic genotypes, providing a high-fidelity modeling platform for skeletal muscle laminopathies. Our results lay the foundation for future iPSC-based therapy development and screening platforms for skeletal muscle laminopathies.

19.
Nucleic Acids Res ; 46(16): 8275-8298, 2018 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-29947794

RESUMEN

CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3'-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Mioblastos/metabolismo , Distrofia Miotónica/genética , Expansión de Repetición de Trinucleótido/genética , Células Cultivadas , Niño , Femenino , Humanos , Persona de Mediana Edad , Desarrollo de Músculos/genética , Distrofia Miotónica/metabolismo , Distrofia Miotónica/patología
20.
Cell Rep ; 23(3): 899-908, 2018 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-29669293

RESUMEN

Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development.


Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Ingeniería de Tejidos , Diferenciación Celular , Linaje de la Célula , Humanos , Hidrogeles/química , Desarrollo de Músculos , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Andamios del Tejido/química
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