RESUMEN
At the neuromuscular junction, the downstream of tyrosine kinase 7 (DOK7) enhances the phosphorylation of muscle-specific kinase (MuSK) and induces clustering of acetylcholine receptors (AChRs). We identified a patient with congenital myasthenic syndrome (CMS) with two heteroallelic mutations in DOK7, c.653-1G>C in intron 5 and c.190G>A predicting p.G64R in the pleckstrin homology domain. iPS cells established from the patient (CMS-iPSCs) showed that c.653-1G>C caused in-frame skipping of exon 6 (120 bp) and frame-shifting activation of a cryptic splice site deleting seven nucleotides in exon 6. p.G64R reduced the expression of DOK7 to 10% of wild-type DOK7, and markedly compromised AChR clustering in transfected C2C12 myotubes. p.G64R-DOK7 made insoluble aggresomes at the juxtanuclear region in transfected C2C12 myoblasts and COS7 cells, which were co-localized with molecules in the autophagosome system. A protease inhibitor MG132 reduced the soluble fraction of p.G64R-DOK7 and enhanced the aggresome formation of p.G64R-DOK7. To match the differentiation levels between patient-derived and control induced pluripotent stem cells (iPSCs), we corrected c.190G>A (p.G64R) by CRISPR/Cas9 to make isogenic iPSCs while retaining c.653-1G>C (CMS-iPSCsCas9). Myogenically differentiated CMS-iPSCs showed juxtanuclear aggregates of DOK7, reduced expression of endogenous DOK7 and reduced phosphorylation of endogenous MuSK. Another mutation, p.T77M, also made aggresome to a less extent compared with p.G64R in transfected COS7 cells. These results suggest that p.G64R-DOK7 makes aggresomes in cultured cells and is likely to compromise MuSK phosphorylation for AChR clustering.
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Células Madre Pluripotentes Inducidas , Síndromes Miasténicos Congénitos , Humanos , Células Cultivadas , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Musculares/genética , Mutación , Síndromes Miasténicos Congénitos/genética , Síndromes Miasténicos Congénitos/metabolismo , Fosforilación , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismoRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is an inherited muscle disease caused by misexpression of the DUX4 gene in skeletal muscle. DUX4 is a transcription factor, which is normally expressed in the cleavage-stage embryo and regulates gene expression involved in early embryonic development. Recent studies revealed that DUX4 also activates the transcription of repetitive elements such as endogenous retroviruses (ERVs), mammalian apparent long terminal repeat (LTR)-retrotransposons and pericentromeric satellite repeats (Human Satellite II). DUX4-bound ERV sequences also create alternative promoters for genes or long non-coding RNAs, producing fusion transcripts. To further understand transcriptional regulation by DUX4, we performed nanopore long-read direct RNA sequencing (dRNA-seq) of human muscle cells induced by DUX4, because long reads show whole isoforms with greater confidence. We successfully detected differential expression of known DUX4-induced genes and discovered 61 differentially expressed repeat loci, which are near DUX4-ChIP peaks. We also identified 247 gene-ERV fusion transcripts, of which 216 were not reported previously. In addition, long-read dRNA-seq clearly shows that RNA splicing is a common event in DUX4-activated ERV transcripts. Long-read analysis showed non-LTR transposons including Alu elements are also transcribed from LTRs. Our findings revealed further complexity of DUX4-induced ERV transcripts. This catalogue of DUX4-activated repetitive elements may provide useful information to elucidate the pathology of FSHD. Also, our results indicate that nanopore dRNA-seq has complementary strengths to conventional short-read complementary DNA sequencing.
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Proteínas de Homeodominio/genética , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapulohumeral/genética , Nanoporos , Secuencias Repetitivas de Ácidos Nucleicos/genética , Análisis de Secuencia de ARN/métodos , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Células Musculares/metabolismo , Distrofia Muscular Facioescapulohumeral/patología , Isoformas de Proteínas/genética , Isoformas de ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN/estadística & datos numéricosRESUMEN
Skeletal muscle mass is negatively regulated by several TGF-ß superfamily members. Myostatin (MSTN) is the most prominent negative regulator of muscle mass. Recent studies show that in addition to MSTN, GDF11, which shares a high sequence identity with MSTN, induces muscle atrophy in vitro and in vivo at supraphysiological levels, whereas controversy regarding its roles exists. Furthermore, higher circulating GDF11 levels associate with frailty in humans. On the other hand, little is known about the effect of pathophysiological levels of GDF11 on muscle atrophy. Here we seek to determine whether pathophysiological levels of GDF11 are sufficient to activate Smad2/Smad3 signaling and induce muscle atrophy using human iPSC-derived myocytes (hiPSC myocytes). We first show that incubating hiPSC myocytes with pathophysiological concentrations of GDF11 significantly reduces myocyte diameters. We next demonstrate that pathophysiological levels of GDF11 are sufficient to activate Smad2/3 signaling. Finally, we show that pathophysiological levels of GDF11 are capable of inducing the expression of Atrogin-1, an atrophy-promoting E3 ubiquitin ligase and that FOXO1 blockage reverses the GDF11-induced Atrogin-1 expression and atrophic phenotype. Collectively, our results suggest that GDF11 induces skeletal muscle atrophy at the pathophysiological levels through the GDF11-FOXO1 axis.
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Células Madre Pluripotentes Inducidas , Miostatina , Humanos , Miostatina/genética , Miostatina/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Factores de Diferenciación de Crecimiento/genética , Factores de Diferenciación de Crecimiento/metabolismo , Factores de Diferenciación de Crecimiento/farmacología , Atrofia Muscular/patología , Músculo Esquelético/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Células Musculares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína smad3/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Proteína Smad2/genéticaRESUMEN
The disruption of excitation-contraction (EC) coupling and subsequent reduction in Ca2+ release from the sarcoplasmic reticulum (SR) have been shown to account for muscle weakness seen in patients with Duchenne muscular dystrophy (DMD). Here, we examined the mechanisms underlying EC uncoupling in skeletal muscles from mdx52 and DMD-null/NSG mice, animal models for DMD, focusing on the SH3 and cysteine-rich domain 3 (STAC3) and junctophilin 1 (JP1), which link the dihydropyridine receptor (DHPR) in the transverse tubule and the ryanodine receptor 1 in the SR. The isometric plantarflexion torque normalized to muscle weight of whole plantar flexor muscles was depressed in mdx52 and DMD-null/NSG mice compared with their control mice. This was accompanied by increased autolysis of calpain-1, decreased levels of STAC3 and JP1 content, and dissociation of STAC3 and JP1 from DHPR-α1s in gastrocnemius muscles. Moreover, in vitro mechanistic experiments demonstrated that STAC3 and JP1 underwent Ca2+-dependent proteolysis that was less pronounced in dystrophin-deficient muscles where calpastatin, the endogenous calpain inhibitor, was upregulated. Eccentric contractions further enhanced autolysis of calpain-1 and proteolysis of STAC3 and JP1 that were associated with severe torque depression in gastrocnemius muscles from DMD-null/NSG mice. These data suggest that Ca2+-dependent proteolysis of STAC3 and JP1 may be an essential factor causing muscle weakness due to EC coupling failure in dystrophin-deficient muscles.NEW & NOTEWORTHY The mechanisms underlying the disruption of excitation-contraction (EC) coupling in dystrophin-deficient muscles are not well understood. Here, using animal models for Duchenne muscular dystrophies (DMD), we show a Ca2+-dependent protease (calpain-1)-mediated proteolysis of SH3 and cysteine-rich domain 3 (STAC3) and junctophilin 1 (JP1), essential EC coupling proteins, in dystrophin-deficient muscle, and highlighting the dissociation of STAC3 and JP1 from dihydropyridine receptor as a causative factor in EC uncoupling of dystrophic muscles.
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Canales de Calcio Tipo L , Distrofia Muscular de Duchenne , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Calpaína/metabolismo , Cisteína/metabolismo , Distrofina/genética , Distrofina/metabolismo , Proteínas de la Membrana , Ratones , Ratones Endogámicos mdx , Debilidad Muscular/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismoRESUMEN
Somites (SMs) comprise a transient stem cell population that gives rise to multiple cell types, including dermatome (D), myotome (MYO), sclerotome (SCL) and syndetome (SYN) cells. Although several groups have reported induction protocols for MYO and SCL from pluripotent stem cells, no studies have demonstrated the induction of SYN and D from SMs. Here, we report systematic induction of these cells from human induced pluripotent stem cells (iPSCs) under chemically defined conditions. We also successfully induced cells with differentiation capacities similar to those of multipotent mesenchymal stromal cells (MSC-like cells) from SMs. To evaluate the usefulness of these protocols, we conducted disease modeling of fibrodysplasia ossificans progressiva (FOP), an inherited disease that is characterized by heterotopic endochondral ossification in soft tissues after birth. Importantly, FOP-iPSC-derived MSC-like cells showed enhanced chondrogenesis, whereas FOP-iPSC-derived SCL did not, possibly recapitulating normal embryonic skeletogenesis in FOP and cell-type specificity of FOP phenotypes. These results demonstrate the usefulness of multipotent SMs for disease modeling and future cell-based therapies.
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Desarrollo Óseo , Condrogénesis , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Miositis Osificante/metabolismo , Somitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/patología , Miositis Osificante/patología , Somitos/patologíaRESUMEN
Double homeobox 4 (DUX4), the causative gene of facioscapulohumeral muscular dystrophy (FSHD), is ectopically expressed in the skeletal muscle cells of FSHD patients because of chromatin relaxation at 4q35. The diminished heterochromatic state at 4q35 is caused by either large genome contractions [FSHD type 1 (FSHD1)] or mutations in genes encoding chromatin regulators, such as SMCHD1 [FSHD type 2 (FSHD2)]. However, the mechanism by which DUX4 expression is regulated remains largely unknown. Here, using a myocyte model developed from patient-derived induced pluripotent stem cells, we determined that DUX4 expression was increased by oxidative stress (OS), a common environmental stress in skeletal muscle, in both FSHD1 and FSHD2 myocytes. We generated FSHD2-derived isogenic control clones with SMCHD1 mutation corrected by clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) and homologous recombination and found in the myocytes obtained from these clones that DUX4 basal expression and the OS-induced upregulation were markedly suppressed due to an increase in the heterochromatic state at 4q35. We further found that DNA damage response (DDR) was involved in OS-induced DUX4 increase and identified ataxia-telangiectasia mutated, a DDR regulator, as a mediator of this effect. Our results suggest that the relaxed chromatin state in FSHD muscle cells permits aberrant access of OS-induced DDR signaling, thus increasing DUX4 expression. These results suggest OS could represent an environmental risk factor that promotes FSHD progression.
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Proteínas Cromosómicas no Histona/genética , Proteínas de Homeodominio/genética , Células Madre Pluripotentes Inducidas/metabolismo , Distrofia Muscular Facioescapulohumeral/genética , Sistemas CRISPR-Cas/genética , Cromatina/genética , Cromosomas Humanos Par 4/genética , Daño del ADN/genética , Regulación de la Expresión Génica , Humanos , Células Musculares/metabolismo , Células Musculares/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular Facioescapulohumeral/metabolismo , Distrofia Muscular Facioescapulohumeral/patología , Mutación , Estrés Oxidativo/genéticaRESUMEN
Skeletal muscle stem cells (MuSCs) have been proposed as suitable candidates for cell therapy in muscular disorders since they exhibit good capacity for myogenic regeneration. However, for better therapeutic outcomes, it is necessary to isolate human MuSCs from a suitable tissue source with high myogenic differentiation. In this context, we isolated CD56+CD82+ cells from the extra eyelid tissue of young and aged patients, and tested in vitro myogenic differentiation potential. In the current study, myogenic cells derived from extra eyelid tissue were characterized and compared with immortalized human myogenic cells. We found that myogenic cells derived from extra eyelid tissue proliferated and differentiated myofibers in vitro, and restored DYSTROPHIN or PAX7 expression after transplantation with these cells in mice with Duchenne muscular dystrophy. Thus, human myogenic cells derived from extra eyelid tissue including the orbicularis oculi might be good candidates for stem cell-based therapies for treating muscular diseases.
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Diferenciación Celular , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Trasplante de Células Madre , Células Madre/citología , Células Madre/metabolismo , Animales , Biomarcadores , Células Cultivadas , Humanos , Inmunofenotipificación , Ratones , Distrofia Muscular de Duchenne/terapia , FenotipoRESUMEN
Mitochondrial oxidative capacity in skeletal muscle is known to decrease in diabetic patients, and sarcopenia is a risk factor for diabetes, particularly in elderly people. We previously revealed that microRNA (miR)-494 inhibits mitochondrial biogenesis during myogenic differentiation in murine C2C12 cells and others reported that exercise regulates miR-494 levels in obese sedentary individuals with increased risk of type 2 diabetes. In this study, to investigate the therapeutic potential of miR-494, we first investigated the role of miR-494 during human skeletal myogenesis. Using human induced pluripotent stem (hiPS) cells stably transfected with the Tet/ON-myogenic differentiation 1(MYOD1) gene (MyoD-hiPS cells), we found that miR-494 expression transiently increased and was downregulated after myogenic induction. In miR-494 transfected MyoD-hiPS cells, the level of high oxidative fiber (type IIa) marker proteins specifically decreased, while no change in the total number of cells was observed. In contrast, the expression of both type I and type IIx markers was unaffected by miR-494 overexpression. Furthermore, miR-494 overexpression suppressed basal oxygen consumption rate concomitant with the inhibition of myotube formation and without significant effects on the mitochondrial content. These results suggest that miR-494 plays a novel role in the fiber type-specific skeletal myogenesis in MyoD-hiPS cells, distinct from murine C2C12 myogenesis.
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Células Madre Pluripotentes Inducidas/metabolismo , MicroARNs/genética , Desarrollo de Músculos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Línea Celular , Regulación hacia Abajo , Regulación del Desarrollo de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , MicroARNs/metabolismo , Mitocondrias Musculares/metabolismo , Fibras Musculares Esqueléticas/citología , Músculo Esquelético/metabolismo , Regulación hacia ArribaRESUMEN
INTRODUCTION: Carnitine palmitoyltransferase II (CPT II) deficiency is an inherited disorder involving ß-oxidation of long-chain fatty acids (FAO), which leads to rhabdomyolysis and subsequent acute renal failure. The detailed mechanisms of disease pathogenesis remain unknown; however, the availability of relevant human cell types for investigation, such as skeletal muscle cells, is limited, and the development of novel disease models is required. METHODS: We generated human induced pluripotent stem cells (hiPSCs) from skin fibroblasts of a Japanese patient with CPT II deficiency. Mature myocytes were differentiated from the patient-derived hiPSCs by introducing myogenic differentiation 1 (MYOD1), the master transcriptional regulator of myocyte differentiation. Using an in vitro acylcarnitine profiling assay, we investigated the effects of a hypolipidemic drug, bezafibrate, and heat stress on mitochondrial FAO in CPT II-deficient myocytes and controls. RESULTS: CPT II-deficient myocytes accumulated more palmitoylcarnitine (C16) than did control myocytes. Heat stress, induced by incubation at 38°C, leads to a robust increase of C16 in CPT II-deficient myocytes, but not in controls. Bezafibrate reduced the amount of C16 in control and CPT II-deficient myocytes. DISCUSSION: In this study, we induced differentiation of CPT II-deficient hiPSCs into mature myocytes in a highly efficient and reproducible manner and recapitulated some aspects of the disease phenotypes of CPT II deficiency in the myocyte disease models. This approach addresses the challenges of modeling the abnormality of FAO in CPT II deficiency using iPSC technology and has the potential to revolutionize translational research in this field.
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Carnitina O-Palmitoiltransferasa/deficiencia , Errores Innatos del Metabolismo/metabolismo , Errores Innatos del Metabolismo/patología , Células Musculares/patología , Células Madre Pluripotentes/patología , Bezafibrato/farmacología , Carnitina/análogos & derivados , Carnitina/metabolismo , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Diferenciación Celular , Células Cultivadas , Fibroblastos/patología , Regulación de la Expresión Génica , Humanos , Masculino , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Palmitoilcarnitina/metabolismo , Células Madre Pluripotentes/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease caused by a gain-of-function mutation in ACVR1, which is a bone morphogenetic protein (BMP) type I receptor. Moreover, it causes progressive heterotopic ossification (HO) in connective tissues. Using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and mouse models, we elucidated the underlying mechanisms of FOP pathogenesis and identified a candidate drug for FOP. METHODS: In the current study, healthy mesenchymal stem/stromal cells derived from iPSCs (iMSCs) expressing ACVR2B-Fc (iMSCACVR2B-Fc), which is a neutralizing receptobody, were constructed. Furthermore, patient-derived iMSCs and FOP mouse model (ACVR1R206H, female) were used to confirm the inhibitory function of ACVR2B-Fc fusion protein secreted by iMSCACVR2B-Fc on BMP signaling pathways and HO development, respectively. RESULTS: We found that secreted ACVR2B-Fc attenuated BMP signaling initiated by Activin-A and BMP-9 in both iMSCs and FOP-iMSCs in vitro. Transplantation of ACVR2B-Fc-expressing iMSCs reduced primary HO in a transgenic mouse model of FOP. Notably, a local injection of ACVR2B-Fc-expressing iMSCs and not an intraperitoneal injection improved the treadmill performance, suggesting compound effects of ACVR2B-Fc and iMSCs. CONCLUSIONS: These results offer a new perspective for treating FOP through stem cell therapy.
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Miositis Osificante , Osificación Heterotópica , Femenino , Humanos , Ratones , Animales , Miositis Osificante/genética , Miositis Osificante/terapia , Osificación Heterotópica/terapia , Osificación Heterotópica/genética , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/farmacología , Transducción de Señal , Ratones Transgénicos , Mutación , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Receptores de Activinas Tipo II/farmacologíaRESUMEN
Human-induced pluripotent stem cells (hiPSCs) have great therapeutic potential. The cell source differentiated from hiPSCs requires xeno-free and robust methods for lineage-specific differentiation. Here, a system is described for differentiating hiPSCs on new generation laminin fragments (NGLFs), a recombinant form of a laminin E8 fragment conjugated to the heparan sulfate chains (HS) attachment domain of perlecan. Using NGLFs, hiPSCs are highly promoted to direct differentiation into a paraxial mesoderm state with high-efficiency muscle lineage generation. HS conjugation to the C-terminus of Laminin E8 fragments brings fibroblast growth factors (FGFs) bound to the HS close to the cell surface of hiPSCs, thereby facilitating stronger FGF signaling pathways stimulation and initiating HOX gene expression, which triggers the paraxial mesoderm differentiation of hiPSCs. This highly efficient differentiation system can provide a roadmap for paraxial mesoderm development and an infinite source of myocytes and muscle stem cells for disease modeling and regenerative medicine.
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Diferenciación Celular , Heparitina Sulfato , Células Madre Pluripotentes Inducidas , Laminina , Mesodermo , Diferenciación Celular/fisiología , Mesodermo/citología , Mesodermo/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Laminina/metabolismo , Heparitina Sulfato/metabolismo , Desarrollo de Músculos/fisiología , Desarrollo de Músculos/genética , Células CultivadasRESUMEN
Ehlers-Danlos syndrome spondylodysplastic type 3 (EDSSPD3, OMIM 612350) is an inherited recessive connective tissue disorder that is caused by loss of function of SLC39A13/ZIP13, a zinc transporter belonging to the Slc39a/ZIP family. We previously reported that patients with EDSSPD3 harboring a homozygous loss of function mutation (c.221G > A, p.G64D) in ZIP13 exon 2 (ZIP13G64D) suffer from impaired development of bone and connective tissues, and muscular hypotonia. However, whether ZIP13 participates in the early differentiation of these cell types remains unclear. In the present study, we investigated the role of ZIP13 in myogenic differentiation using a murine myoblast cell line (C2C12) as well as patient-derived induced pluripotent stem cells (iPSCs). We found that ZIP13 gene expression was upregulated by myogenic stimulation in C2C12 cells, and its knockdown disrupted myotubular differentiation. Myocytes differentiated from iPSCs derived from patients with EDSSPD3 (EDSSPD3-iPSCs) also exhibited incomplete myogenic differentiation. Such phenotypic abnormalities of EDSSPD3-iPSC-derived myocytes were corrected by genomic editing of the pathogenic ZIP13G64D mutation. Collectively, our findings suggest the possible involvement of ZIP13 in myogenic differentiation, and that EDSSPD3-iPSCs established herein may be a promising tool to study the molecular basis underlying the clinical features caused by loss of ZIP13 function.
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Proteínas Portadoras , Síndrome de Ehlers-Danlos , Osteocondrodisplasias , Animales , Humanos , Ratones , Diferenciación Celular/genéticaRESUMEN
For cell therapy toward Duchenne muscle dystrophy (DMD), muscle progenitor cells derived from human-induced pluripotent stem cell (hiPSC-MuPCs) are recognized as a good candidate, and currently, cell transplantation of hiPSC-MuPCs is being tested with several DMD animal models. In this article, we describe an efficient method to dissociate, purify by cell sorting, transplant, and evaluate the transplantation efficacy of hiPSC-MuPCs.
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Células Madre Pluripotentes Inducidas , Distrofia Muscular de Duchenne , Ratones , Humanos , Animales , Distrofia Muscular de Duchenne/terapia , Mioblastos , Trasplante de Células Madre , Modelos Animales de Enfermedad , MúsculosRESUMEN
Application of the tetracycline-inducible gene expression system (Tet-On) in human induced pluripotent stem cells (hiPSCs) has become a fundamental transgenic tool owing to its regulatable gene expression. One of the major hurdles in hiPSC application is non-uniform transgene activation. Here, we report that the supplementation of reverse tetracycline transactivator (rtTA) in polyclonal hiPSCs populations can achieve the uniform transgene activation of Tet-On. Furthermore, the choice of antibiotic selection markers connected by an internal ribosomal entry site (IRES) can influence the expression of upstream transgenes. In particular, expression of the rtTA is more uniform in cell populations when linked to puromycin as compared to neomycin, obviating the need for sub-cloning or supplementation of rtTA. Finally, to expand the range of applications, we adopted our findings to tetracycline-inducible MyoD vector (Tet-MyoD). Our Tet-MyoD promises efficient, robust, and reproducible directed myogenic differentiation of hiPSCs.
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Most muscular dystrophies are the result of genetic disorders. There is currently no effective treatment for these progressive diseases except palliative therapy. Muscle stem cells with potent self-renewal and regenerative potential are considered a target for treating muscular dystrophy. Human induced pluripotent stem cells have been expected as a source of MuSCs because of their infinite proliferation potential and less immunogenicity. However, the generation of engraftable MuSCs from hiPSCs is relatively difficult and encounters low efficiency and reproducibility. Here, we introduce a transgene-free protocol of hiPSCs differentiating into fetal MuSCs by identifying them as MYF5-positive cells. Flow cytometry analysis detected around 10% of MYF5-positive cells after 12 weeks of differentiation. Approximately 50 ~ 60% of MYF5-positive cells were positively identified using Pax7 immunostaining. This differentiation protocol is expected to be useful for not only the establishment of cell therapy but also the future drug discovery using patient-derived hiPSCs.
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Células Madre Pluripotentes Inducidas , Distrofias Musculares , Humanos , Reproducibilidad de los Resultados , Células Cultivadas , Diferenciación Celular , Fibras Musculares Esqueléticas , Músculo EsqueléticoRESUMEN
Duchenne muscular dystrophy (DMD) is an X-linked fatal muscular disease, affecting one in 3,500 live male births worldwide. Currently, there is no cure for this disease, except for steroid-based treatment to attenuate disease progression. Cell transplantation therapy is a promising therapeutic approach, however, there is a lack of appropriate animal models to conduct large-scale preclinical studies using human cells, including biochemical and functional tests. Here, we established an immunodeficient DMD rat model and performed exhaustive pathological analysis and transplantation efficiency evaluation to assess its suitability to study DMD. Our DMD rat model exhibited histopathological characteristics similar to those observed in human patients with DMD. Human myoblasts demonstrated successful engraftment following transplantation into these rats. Therefore, this immunodeficient DMD rat model would be useful in preclinical studies to develop cellular transplantation therapies for DMD.
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Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats (CTGexp) in the dystrophia myotonica protein kinase (DMPK) gene, and the transcription products, expanded CUG repeats, sequester muscleblind like splicing regulator 1 (MBNL1), resulting in the nuclear MBNL1 aggregation in the DM1 cells. Loss of MBNL1 function is the pivotal mechanism underlying the pathogenesis of DM1. To develop therapeutics for DM1, proper human in vitro models based on the pathologic mechanism of DM1 are required. In this study, we established robust in vitro skeletal muscle cell models of DM1 with patient-derived induced pluripotent stem cells (iPSCs) using the MyoD1-induced system and iPSCs-derived muscle stem cell (iMuSC) differentiation system. Our newly established DM1 models enable simple quantitative evaluation of nuclear MBNL1 aggregation and the downstream splicing defects. Quantitative analyses using the MyoD1-induced myotubes showed that CTGexp-deleted DM1 skeletal myotubes exhibited a reversal of MBNL1-related pathologies, and antisense oligonucleotide treatment recovered these disease phenotypes in the DM1-iPSCs-derived myotubes. Furthermore, iMuSC-derived myotubes exhibited higher maturity than the MyoD1-induced myotubes, which enabled us to recapitulate the SERCA1 splicing defect in the DM1-iMuSC-derived myotubes. Our quantitative and reproducible in vitro models for DM1 established using human iPSCs are promising for drug discovery against DM1.
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Células Madre Pluripotentes Inducidas , Distrofia Miotónica , Humanos , Empalme Alternativo , Células Madre Pluripotentes Inducidas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Miotónica/patología , Empalme del ARN , Modelos BiológicosRESUMEN
Schwartz-Jampel syndrome (SJS) is an autosomal recessive disorder caused by loss-of-function mutations in heparan sulfate proteoglycan 2 (HSPG2), which encodes the core basement membrane protein perlecan. Myotonia is a major criterion for the diagnosis of SJS; however, its evaluation is based solely on physical examination and can be challenging in neonates and young children. Furthermore, the pathomechanism underlying SJS-related myotonia is not fully understood, and effective treatments for SJS are limited. Here, we established a cellular model of SJS using patient-derived human-induced pluripotent stem cells. This model exhibited hyper-responsiveness to acetylcholine as a result of abnormalities in the perlecan molecule, which were confirmed via comparison of their calcium imaging with calcium imaging of satellite cells derived from Hspg2-/--Tg mice, which exhibit myotonic symptoms similar to SJS symptoms. Therefore, our results confirm the utility of creating cellular models for investigating SJS and their application in evaluating myotonia in clinical cases, while also providing a useful tool for the future screening of SJS therapies.
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To restore dystrophin protein in various mutation patterns of Duchenne muscular dystrophy (DMD), the multi-exon skipping (MES) approach has been investigated. However, only limited techniques are available to induce a large deletion to cover the target exons spread over several hundred kilobases. Here, we utilized the CRISPR-Cas3 system for MES induction and showed that dual crRNAs could induce a large deletion at the dystrophin exon 45-55 region (â¼340 kb), which can be applied to various types of DMD patients. We developed a two-color SSA-based reporter system for Cas3 to enrich the genome-edited cell population and demonstrated that MES induction restored dystrophin protein in DMD-iPSCs with three distinct mutations. Whole-genome sequencing and distance analysis detected no significant off-target deletion near the putative crRNA binding sites. Altogether, dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein via MES induction.
Asunto(s)
Distrofina , Distrofia Muscular de Duchenne , Humanos , Distrofina/genética , Sistemas CRISPR-Cas/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Sitios de Unión , Exones/genéticaRESUMEN
Due to their physiological relevance, cell-based assays using human-induced pluripotent stem cell (iPSC)-derived cells are a promising in vitro pharmacological evaluation system for drug candidates. However, cell-based assays involve complex processes such as long-term culture, real-time and continuous observation of living cells, and detection of many cellular events. Automating multi-sample processing through these assays will enhance reproducibility by limiting human error and reduce researchers' valuable time spent conducting these experiments. Furthermore, this integration enables continuous tracking of morphological changes, which is not possible with the use of stand-alone devices. This report describes a new laboratory automation system called the Screening Station, which uses novel automation control and scheduling software called Green Button Go to integrate various devices. To integrate the above-mentioned processes, we established three workflows in Green Button Go: 1) For long-term cell culture, culture plates and medium containers are transported from the automatic CO2 incubator and cool incubator, respectively, and the cell culture medium in the microplates is exchanged daily using the Biomek i7 workstation; 2) For time-lapse live-cell imaging, culture plates are automatically transferred between the CQ1 confocal quantitative image cytometer and the SCALE48W automatic CO2 incubator; 3) For immunofluorescence imaging assays, in addition to the above-mentioned devices, the 405LS microplate washer allows for formalin-fixation and immunostaining of cells. By scheduling various combinations of the three workflows, we successfully automated the culture and medium exchange processes for iPSCs derived from patients with facioscapulohumeral muscular dystrophy, confirmation of their differentiation status by live-cell imaging, and confirmation of the presence of differentiation markers by immunostaining. In addition, deep learning analysis enabled us to quantify the degree of iPSC differentiation from live-cell imaging data. Further, the results of the fully automated experiments could be accessed via the intranet, enabling experiments and analysis to be conducted remotely once the necessary reagents and labware were prepared. We expect that the ability to perform clinically and physiologically relevant cell-based assays from remote locations using the Screening Station will facilitate global research collaboration and accelerate the discovery of new drug candidates.