Skeletal muscle research is transitioning toward 3D tissue engineered in vitro models reproducing muscle's native architecture and supporting measurement of functionality. Human induced pluripotent... Show moreSkeletal muscle research is transitioning toward 3D tissue engineered in vitro models reproducing muscle's native architecture and supporting measurement of functionality. Human induced pluripotent stem cells (hiPSCs) offer high yields of cells for differentiation. It has been difficult to differentiate high-quality, pure 3D muscle tissues from hiPSCs that show contractile properties comparable to primary myoblast-derived tissues. Here, we present a transgene-free method for the generation of purified, expandable myogenic progenitors (MPs) from hiPSCs grown under feeder-free conditions. We defined a protocol with optimal hydrogel and medium conditions that allowed production of highly contractile 3D tissue engineered skeletal muscles with forces similar to primary myoblast-derived tissues. Gene expression and proteomic analysis between hiPSC-derived and primary myoblast-derived 3D tissues revealed a similar expression profile of proteins involved in myogenic differentiation and sarcomere function. The protocol should be generally applicable for the study of personalized human skeletal muscle tissue in health and disease. Show less
Iuliano, A.; Haalstra, M.; Raghuraman, R.; Bielawski, K.; Bholasing, A.P.; Wal, E. van der; ... ; Pijnappel, W.W.M.P. 2023
As the field of cardiac and skeletal muscle tissue engineering expands, so does the need for accurate and reliable systems to generate in vitro 3D tissues and analyze their functional properties.... Show moreAs the field of cardiac and skeletal muscle tissue engineering expands, so does the need for accurate and reliable systems to generate in vitro 3D tissues and analyze their functional properties. In this study, the Cuore is introduced, a system that integrates sensors based on optical fibers and uses the principle of light interferometry to detect the contraction of 3D Tissue Engineered Skeletal Muscles (3D-TESMs). The technology employed in the Cuore allows for reproducible and multichannel force measurements down to a nano-Newtons resolution while maintaining sterility and permitting continuous non-invasive recording within and outside standard tissue culture incubators. Thanks to the integrated electrodes for electrical pulse stimulation (EPS), 3D-TESMs generated from three independent hiPSC-derived myogenic progenitors (MPs) lines are stimulated and the contractility is recorded over the course of a week. Through the modulation of different EPS parameters, the optimal combination to induce the 3D-TESMs in producing fully fused tetani without causing damage is determined. Furthermore, 3D-TESMs from different lines exhibit characteristic signatures of spontaneous contractility and response to caffeine, verapamil, and the & beta;-agonist clenbuterol. The ease of use, high sensitivity, and the integrated electrodes and sensors make the Cuore an ideal technology to investigate the biology of contractile tissues and their response to drugs.The expanding field of cardiac and skeletal muscle tissue engineering demands efficient solutions to study the biology of the tissues and model disorders. In this study the Cuore system is presented, employing optical fiber sensors and light interferometry to detect 3D Tissue Engineered Skeletal Muscle contractions. Thanks to nano-Newton resolution force measurements and integrated electrical pulse stimulation, contractility of hiPSC-derived tissues is evaluated. Additionally, its efficacy in assessing drug responses, like caffeine and clenbuterol, showcases its potential in studying contractile tissue biology.image Show less
The interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its... Show moreThe interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its silencing capacity at endogenous developmental targets. Moreover, it also results in enhanced silencing at the facioscapulohumeral muscular dystrophy associated macrosatellite-array, D4Z4, resulting in enhanced repression of DUX4 encoded by this repeat. Heightened SMCHD1 silencing perturbs developmental Hox gene activation, causing a homeotic transformation in mice. Paradoxically, the mutant SMCHD1 appears to enhance insulation against other epigenetic regulators, including PRC2 and CTCF, while depleting long range chromatin interactions akin to what is observed in the absence of SMCHD1. These data suggest that SMCHD1's role in long range chromatin interactions is not directly linked to gene silencing or insulating the chromatin, refining the model for how the different levels of SMCHD1-mediated chromatin regulation interact to bring about gene silencing in normal development and disease.Here the authors reveal that a neomorphic mutation in chromatin protein SMCHD1 enhances SMCHD1-mediated gene silencing, including at the FSHD disease-relevant locus, while depleting SMCHD1-mediated chromatin interactions, suggesting these SMCHD1 functions are unlinked. Show less
The ubiquitin proteasomal system is a critical regulator of muscle physiology, and impaired UPS is key in many muscle pathologies. Yet, little is known about the function of deubiquitinating... Show moreThe ubiquitin proteasomal system is a critical regulator of muscle physiology, and impaired UPS is key in many muscle pathologies. Yet, little is known about the function of deubiquitinating enzymes (DUBs) in the muscle cell context. We performed a genetic screen to identify DUBs as potential regulators of muscle cell differentiation. Surprisingly, we observed that the depletion of ubiquitin-specific protease 18 (USP18) affected the differentiation of muscle cells. USP18 depletion first stimulated differentiation initiation. Later, during differentiation, the absence of USP18 expression abrogated myotube maintenance. USP18 enzymatic function typically attenuates the immune response by removing interferon-stimulated gene 15 (ISG15) from protein substrates. However, in muscle cells, we found that USP18, predominantly nuclear, regulates differentiation independent of ISG15 and the ISG response. Exploring the pattern of RNA expression profiles and protein networks whose levels depend on USP18 expression, we found that differentiation initiation was concomitant with reduced expression of the cell-cycle gene network and altered expression of myogenic transcription (co) factors. We show that USP18 depletion altered the calcium channel gene network, resulting in reduced calcium flux in myotubes. Additionally, we show that reduced expression of sarcomeric proteins in the USP18 proteome was consistent with reduced contractile force in an engineered muscle model. Our results revealed nuclear USP18 as a critical regulator of differentiation initiation and maintenance, independent of ISG15 and its role in the ISG response. Show less
Facioscapulohumeral muscular dystrophy (FSHD) is the second most common genetic myopathy, characterized by slowly progressing and highly heterogeneous muscle wasting with a typical onset in the... Show moreFacioscapulohumeral muscular dystrophy (FSHD) is the second most common genetic myopathy, characterized by slowly progressing and highly heterogeneous muscle wasting with a typical onset in the late teens/early adulthood [1]. Although the etiology of the disease for both FSHD type 1 and type 2 has been attributed to gain-of-toxic function stemming from aberrant DUX4 expression, the exact pathogenic mechanisms involved in muscle wasting have yet to be elucidated [2-4]. The 2021 FSHD International Research Congress, held virtually on June 24-25, convened over 350 researchers and clinicians to share the most recent advances in the understanding of the disease mechanism, discuss the proliferation of interventional strategies and refinement of clinical outcome measures, including results from the ReDUX4 trial, a phase 2b clinical trial of losmapimod in FSHD [NCT04003974]. Show less
Bouwman, L.F.; Hamer, B. den; Heuvel, A. van den; Franken, M.; Jackson, M.; Dwyer, C.A.; ... ; Greef, J.C. de 2021
Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent skeletal muscle dystrophies. Skeletal muscle pathology in individuals with FSHD is caused by inappropriate expression of... Show moreFacioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent skeletal muscle dystrophies. Skeletal muscle pathology in individuals with FSHD is caused by inappropriate expression of the transcription factor DUX4, which activates different myotoxic pathways. At the moment there is no molecular therapy that can delay or prevent skeletal muscle wasting in FSHD. In this study, a systemically delivered antisense oligonucleotide (ASO) targeting the DUX4 transcript was tested in vivo in ACTA1-MCM;FLExDUX4 mice that express DUX4 in skeletal muscles. We show that the DUX4 ASO was well tolerated and repressed the DUX4 transcript, DUX4 protein, and mouse DUX4 target gene expression in skeletal muscles. In addition, the DUX4 ASO alleviated the severity of skeletal muscle pathology and partially prevented the dysregulation of inflammatory and extracellular matrix genes. DUX4 ASOtreated ACTA1-MCM;FLExDUX4 mice performed better on a treadmill; however, the hanging grid and four-limb grip strength tests were not improved compared to control ASOtreated ACTA1-MCM;FLExDUX4 mice. This study shows that systemic delivery of ASOs targeting DUX4 is a promising therapeutic strategy for FSHD and strategies that further improve the ASO efficacy in skeletal muscle are warranted. Show less
Olie, C.S.; Wal, E. van der; Domagoj, C.; Maton, L.; Greef, J.C. de; Lin, I.H.; ... ; Raz, V. 2020
Muscle wasting and atrophy are regulated by multiple molecular processes, including mRNA processing. Reduced levels of the polyadenylation binding protein nucleus 1 (PABPN1), a multifactorial... Show moreMuscle wasting and atrophy are regulated by multiple molecular processes, including mRNA processing. Reduced levels of the polyadenylation binding protein nucleus 1 (PABPN1), a multifactorial regulator of mRNA processing, cause muscle atrophy. A proteomic study in muscles with reduced PABPN1 levels suggested dysregulation of sarcomeric and cytoskeletal proteins. Here we investigated the hypothesis that reduced PABPN1 levels lead to an aberrant organization of the cytoskeleton. MURC, a plasma membrane-associated protein, was found to be more abundant in muscles with reduced PABPN1 levels, and it was found to be expressed at regions showing regeneration. A polarized cytoskeletal organization is typical for muscle cells, but muscle cells with reduced PABPN1 levels (named as shPAB) were characterized by a disorganized cytoskeleton that lacked polarization. Moreover, cell mechanical features and myogenic differentiation were significantly reduced in shPAB cells. Importantly, restoring cytoskeletal stability, by actin overexpression, was beneficial for myogenesis, expression of sarcomeric proteins and proper localization of MURC in shPAB cell cultures and in shPAB muscle bundle. We suggest that poor cytoskeletal mechanical features are caused by altered expression levels of cytoskeletal proteins and contribute to muscle wasting and atrophy. Show less
Bouwman, L.F.; Maarel, S.M. van der; Greef, J.C. de 2020
Purpose of review Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder, which is caused by incomplete repression of the transcription factor double homeobox 4 (DUX4) in... Show morePurpose of review Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder, which is caused by incomplete repression of the transcription factor double homeobox 4 (DUX4) in skeletal muscle. To date, there is no DUX4-targeting treatment to prevent or delay disease progression. In the present review, we summarize developments in therapeutic strategies with the focus on inhibiting DUX4 and DUX4 target gene expression. Recent findings Different studies show that DUX4 and its target genes can be repressed with genetic therapies using diverse strategies. Additionally, different small compounds can reduce DUX4 and its target genes in vitro and in vivo. Most studies that show DUX4 repression by genetic therapies have only been tested in vitro. More efforts should be made to test them in vivo for clinical translation. Several compounds have been shown to prevent DUX4 and target gene expression in vitro and in vivo. However, their efficiency and specificity has not yet been shown. With emerging clinical trials, the clinical benefit from DUX4 repression in FSHD will likely soon become apparent. Show less
Bouwman, L.F.; Hamer, B. den; Verveer, E.P.; Lerink, L.J.S.; Krom, Y.D.; Maarel, S.M. van der; Greef, J.C. de 2020
Background Facioscapulohumeral muscular dystrophy (FSHD) is a skeletal muscle disorder that is caused by derepression of the transcription factor DUX4 in skeletal muscle cells. Apart from SMCHD1,... Show moreBackground Facioscapulohumeral muscular dystrophy (FSHD) is a skeletal muscle disorder that is caused by derepression of the transcription factor DUX4 in skeletal muscle cells. Apart from SMCHD1, DNMT3B was recently identified as a disease gene and disease modifier in FSHD. However, the exact role of DNMT3B at the D4Z4 repeat array remains unknown. Methods To determine the role of Dnmt3b on DUX4 repression, hemizygous mice with a FSHD-sized D4Z4 repeat array (D4Z4-2.5 mice) were cross-bred with mice carrying an in-frame exon skipping mutation inDnmt3b(Dnmt3b(MommeD14)mice). Additionally, siRNA knockdowns ofDnmt3bwere performed in mouse embryonic stem cells (mESCs) derived from the D4Z4-2.5 mouse model. Results In mESCs derived from D4Z4-2.5 mice, Dnmt3b was enriched at the D4Z4 repeat array and DUX4 transcript levels were upregulated after a knockdown ofDnmt3b. In D4Z4-2.5/Dnmt3b(MommeD14)mice, Dnmt3b protein levels were reduced; however, DUX4 RNA levels in skeletal muscles were not enhanced and no pathology was observed. Interestingly, D4Z4-2.5/Dnmt3b(MommeD14)mice showed a loss of DNA methylation at the D4Z4 repeat array and significantly higher DUX4 transcript levels in secondary lymphoid organs. As these lymphoid organs seem to be more sensitive to epigenetic modifiers of the D4Z4 repeat array, different immune cell populations were quantified in the spleen and inguinal lymph nodes of D4Z4-2.5 mice crossed with Dnmt3b(MommeD14)mice or Smchd1(MommeD1)mice. Only in D4Z4-2.5/Smchd1(MommeD1)mice the immune cell populations were disturbed. Conclusions Our data demonstrates that loss of Dnmt3b results in derepression of DUX4 in lymphoid tissues and mESCs but not in myogenic cells of D4Z4-2.5/Dnmt3b(MommeD14)mice. In addition, the Smchd1(MommeD1)variant seems to have a more potent role in DUX4 derepression. Our studies suggest that the immune system is particularly but differentially sensitive to D4Z4 chromatin modifiers which may provide a molecular basis for the yet underexplored immune involvement in FSHD. Show less
Iuliano, A.; Wal, E. van der; Ruijmbeek, C.W.B.; in't Groen, S.L.M.; Pijnappel, W.W.M.P.; Greef, J.C. de; Saggiomo, V. 2020
The transition from 2D to 3D engineered tissue cultures is changing the way biologists can perform in vitro functional studies. However, there has been a paucity in the establishment of methods... Show moreThe transition from 2D to 3D engineered tissue cultures is changing the way biologists can perform in vitro functional studies. However, there has been a paucity in the establishment of methods required for the generation of microdevices and cost-effective scaling up. To date, approaches including multistep photolithography, milling and 3D printing have been used that involve specialized and expensive equipment or time-consuming steps with variable success. Here, a fabrication pipeline is presented based on affordable off-the-shelf 3D printers and novel replica molding strategies for rapid and easy in-house production of hundreds of 3D culture devices per day, with customizable size and geometry. This pipeline is applied to generate tissue engineered skeletal muscles in vitro using human induced pluripotent stem cell-derived myogenic progenitors. These production methods can be employed in any standard biomedical laboratory. Show less
Muscular dystrophies (MDs) encompass a wide variety of inherited disorders that are characterized by loss of muscle tissue associated with a progressive reduction in muscle function. With a cure... Show moreMuscular dystrophies (MDs) encompass a wide variety of inherited disorders that are characterized by loss of muscle tissue associated with a progressive reduction in muscle function. With a cure lacking for MDs, preclinical developments of therapeutic approaches depend on well-characterized animal models that recapitulate the specific pathology in patients. The mouse is the most widely and extensively used model for MDs, and it has played a key role in our understanding of the molecular mechanisms underlying MD pathogenesis. This has enabled the development of therapeutic strategies. Owing to advancements in genetic engineering, a wide variety of mouse models are available for the majority of MDs. Here, we summarize the characteristics of the most commonly used mouse models for a subset of highly studied MDs, collated into a table. Together with references to key publications describing these models, this brief but detailed overview would be useful for those interested in, or working with, mouse models of MD. Show less
Wal, E. van der; Hamer, B. den; Vliet, P.J. van der; Tok, M.; Brands, T.; Eussen, B.; ... ; Greef, J.C. de 2019
Facioscapulohumeral dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array on chromosome 4q resulting in sporadic misexpression of the transcription factor DUX4 in skeletal... Show moreFacioscapulohumeral dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array on chromosome 4q resulting in sporadic misexpression of the transcription factor DUX4 in skeletal muscle tissue. In similar to 4% of families, de novo D4Z4 contractions occur after fertilization resulting in somatic mosaicism with control and FSHD1 cell populations present within the same patient. Reprogramming of mosaic fibroblasts from two FSHD1 patients into human induced pluripotent stem cells (hiPSCs) generated genetically matched control and FSHD1 hiPSC lines. All hiPSC lines contained a normal karyotype, expressed pluripotency genes and differentiated into cells from the three germ layers. Show less
Greef, J.C. de; Krom, Y.D.; Hamer, B. den; Snider, L.; Hiramuki, Y.; Akker, R.F.P. van den; ... ; Maarel, S.M. van der 2018
Autosomal-recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is mainly characterized by recurrent, often fatal, respiratory and gastrointestinal infections.... Show moreAutosomal-recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is mainly characterized by recurrent, often fatal, respiratory and gastrointestinal infections. About 50% of patients carry mutations in the DNA methyltransferase 3B gene (DNMT3B) (ICF1). The remaining patients carry unknown genetic defects (ICF2) but share with ICF1 patients the same immunological and epigenetic features, including hypomethylation of juxtacentromeric repeat sequences. We performed homozygosity mapping in five unrelated ICF2 patients with consanguineous parents and then performed whole-exome sequencing in one of these patients and Sanger sequencing in all to identify mutations in the zinc-finger- and BTB (bric-a-bric, tramtrack, broad complex)-domain-containing 24 (ZBTB24) gene in four consanguineously descended ICF2 patients. Additionally, we found ZBTB24 mutations in an affected sibling pair and in one patient for whom it was not known whether his parents were consanguineous. ZBTB24 belongs to a large family of transcriptional repressors that include members, such as BCL6 and PATZ1, with prominent regulatory roles in hematopoietic development and malignancy. These data thus indicate that ZBTB24 is involved in DNA methylation of juxtacentromeric DNA and in B cell development and/or B and T cell interactions. Because ZBTB24 is a putative DNA-binding protein highly expressed in the lymphoid lineage, we predict that by studying the molecular function of ZBTB24, we will improve our understanding of the molecular pathophysiology of ICF syndrome and of lymphocyte biology in general. Show less
Autosomal-recessive immunodeficiency, centromeric instability, and facial anomalies (ICE) syndrome is mainly characterized by recurrent, often fatal, respiratory and gastrointestinal infections.... Show moreAutosomal-recessive immunodeficiency, centromeric instability, and facial anomalies (ICE) syndrome is mainly characterized by recurrent, often fatal, respiratory and gastrointestinal infections. About 50% of patients carry mutations in the DNA methyltransferase 3B gene (DNMT3B) (ICF1). The remaining patients carry unknown genetic defects (1072) but share with ICF1 patients the same immunological and epigenetic features, including hypomethylation of juxtacentromeric repeat sequences. We performed homozygosity mapping in five unrelated ICF2 patients with consanguineous parents and then performed whole-exome sequencing in one of these patients and Sanger sequencing in all to identify mutations in the zinc-finger- and BTB (bric-a-bric, tramtrack, broad complex)-domain-containing 24 (ZBTB24) gene in four consanguineously descended ICF2 patients. Additionally, we found ZBTB24 mutations in an affected sibling pair and in one patient for whom it was not known whether his parents were consanguineous. ZBTB24 belongs to a large family of transcriptional repressors that include members, such as BCL6 and PATZ1, with prominent regulatory roles in hematopoietic development and malignancy. These data thus indicate that ZBTB24 is involved in DNA methylation of juxtacentromeric DNA and in B cell development and/or B and cell interactions. Because ZBTB24 is a putative DNA-binding protein highly expressed in the lymphoid lineage, we predict that by studying the molecular function of ZBTB24, we will improve our understanding of the molecular pathophysiology of ICF syndrome and of lymphocyte biology in general. Show less
Greef, J.C. de; Lemmers, R.J.L.F.; Camano, P.; Day, J.W.; Sacconi, S.; Dunand, M.; ... ; Tawil, R. 2010
Objective: In some 5% of patients with facioscapulohumeral muscular dystrophy (FSHD), no D4Z4 repeat contraction on chromosome 4q35 is observed. Such patients, termed patients with FSHD2, show loss... Show moreObjective: In some 5% of patients with facioscapulohumeral muscular dystrophy (FSHD), no D4Z4 repeat contraction on chromosome 4q35 is observed. Such patients, termed patients with FSHD2, show loss of DNA methylation and heterochromatin markers at the D4Z4 repeat that are similar to patients with D4Z4 contractions (FSHD1). This commonality suggests that a change in D4Z4 chromatin structure unifies FSHD1 and FSHD2. The aim of our study was to critically evaluate the clinical features in patients with FSHD2 in order to establish whether these patients are phenotypically identical to FSHD1 and to establish the effects of the (epi-) genotype on the phenotype. Methods: This cross-sectional study studied 33 patients with FSHD2 from 27 families, the largest cohort described to date. All patients were clinically assessed using a standardized clinical evaluation form. Genotype analysis was performed by pulsed field gel electrophoresis and PCR; D4Z4 methylation was studied by methylation-sensitive Southern blot analysis. Results: FSHD2 is identical to FSHD1 in its clinical presentation. Notable differences include a higher incidence (67%) of sporadic cases and the absence of gender differences in disease severity in FSHD2. Overall, average disease severity in FSHD2 was similar to that reported in FSHD1 and was not influenced by D4Z4 repeat size. In FSHD2, a small effect of the degree of hypomethylation on disease severity was observed. Conclusions: Clinically, patients with FSHD2 are indistinguishable from patients with FSHD1. The present data suggest that FSHD1 and FSHD2 are the result of the same pathophysiologic process. Neurology (R) 2010;75:1548-1554 Show less
