De novo revertant fiber formation and therapy testing in a 3D culture model of Duchenne muscular dystrophy skeletal muscle

Ebrahimi, Majid; Lad, Heta; Fusto, Aurora; Tiper, Yekaterina; Datye, Asiman; Nguyen, Christine T. O.; Jacques, Eric; Moyle, Louise A.; Nguyen, T M; Musgrave, Brennen; Chávez-Madero, Carolina; Bigot, Anne; Chen, Chun; Turner, Scott; Stewart, Bryan A.; Pegoraro, Elena; Vitiello, Libero; Gilbert, Penney M.

doi:10.1016/j.actbio.2021.05.020

Cited by 33 publications

(58 citation statements)

References 68 publications

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“…Of note, however, iDRMs require less time, cost, and expertise to generate compared to iPSC-derived myoblasts, which may be especially beneficial for generating patient-specific muscle tissues in time-sensitive or resource-limited settings. Extending culture time ( Santoso and McCain, 2021 ), integrating supporting cell types ( Juhas et al, 2018 ; Santosa et al, 2018 ; Santoso and McCain, 2021 ), providing electrical ( Nedachi et al, 2008 ; Chen et al, 2021 ) or mechanical stimulation ( Heher et al, 2015 ; Chang et al, 2016 ), or engineering 3-D tissues ( Madden et al, 2015 ; Uzel et al, 2016 ; Costantini et al, 2017 ; Davis et al, 2019 ; Ariyasinghe et al, 2020 ; Ebrahimi et al, 2021 ) or earlier exposure to AO could also help induce muscle maturation and proper localization of dystrophin and α-actinin.…”

Section: Discussionmentioning

confidence: 99%

“…Three-dimensional (3-D) muscle tissues have also been engineered by mixing myoblasts in matrix-derived hydrogels and allowing the tissue to compact and align across two anchor points, providing a closer match to the compliance of native tissue and a more relevant microenvironment. 3-D tissues engineered with DMD iPSC-derived myoblasts have demonstrated nuclear, cytoskeletal, and contractile abnormalities ( Maffioletti et al, 2018 ; Ebrahimi et al, 2021 ). However, 3-D tissues models require high numbers of cells and generally entail complex fabrication procedures, limiting their overall throughput.…”

Section: Introductionmentioning

confidence: 99%

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Modeling Patient-Specific Muscular Dystrophy Phenotypes and Therapeutic Responses in Reprogrammed Myotubes Engineered on Micromolded Gelatin Hydrogels

Barthélémy

Santoso

Rabichow

et al. 2022

Front. Cell Dev. Biol.

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In vitro models of patient-derived muscle allow for more efficient development of genetic medicines for the muscular dystrophies, which often present mutation-specific pathologies. One popular strategy to generate patient-specific myotubes involves reprogramming dermal fibroblasts to a muscle lineage through MyoD induction. However, creating physiologically relevant, reproducible tissues exhibiting multinucleated, aligned myotubes with organized striations is dependent on the introduction of physicochemical cues that mimic the native muscle microenvironment. Here, we engineered patient-specific control and dystrophic muscle tissues in vitro by culturing and differentiating MyoD–directly reprogrammed fibroblasts isolated from one healthy control subject, three patients with Duchenne muscular dystrophy (DMD), and two Limb Girdle 2A/R1 (LGMD2A/R1) patients on micromolded gelatin hydrogels. Engineered DMD and LGMD2A/R1 tissues demonstrated varying levels of defects in α-actinin expression and organization relative to control, depending on the mutation. In genetically relevant DMD tissues amenable to mRNA reframing by targeting exon 44 or 45 exclusion, exposure to exon skipping antisense oligonucleotides modestly increased myotube coverage and alignment and rescued dystrophin protein expression. These findings highlight the value of engineered culture substrates in guiding the organization of reprogrammed patient fibroblasts into aligned muscle tissues, thereby extending their value as tools for exploration and dissection of the cellular and molecular basis of genetic muscle defects, rescue, and repair.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Modeling Patient-Specific Muscular Dystrophy Phenotypes and Therapeutic Responses in Reprogrammed Myotubes Engineered on Micromolded Gelatin Hydrogels

Barthélémy

Santoso

Rabichow

et al. 2022

Front. Cell Dev. Biol.

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“…In this case, most works consider the whole area of the engineered tissue (total CSA). However, non-contractile areas can account for up to 90% of the whole CSA ( Sakar et al, 2012 ; Juhas and Bursac, 2014 ; Ebrahimi et al, 2021 ), which has a substantial impact on sF calculations. Thus, other studies consider only the area occupied by the myotubes, which is referred to as effective-CSA ( Hinds et al, 2011 ; Sakar et al, 2012 ; Sato et al, 2013 ; Ebrahimi et al, 2021 ).…”

Section: Contractile Force Analysismentioning

confidence: 99%

“…However, non-contractile areas can account for up to 90% of the whole CSA ( Sakar et al, 2012 ; Juhas and Bursac, 2014 ; Ebrahimi et al, 2021 ), which has a substantial impact on sF calculations. Thus, other studies consider only the area occupied by the myotubes, which is referred to as effective-CSA ( Hinds et al, 2011 ; Sakar et al, 2012 ; Sato et al, 2013 ; Ebrahimi et al, 2021 ). Other authors normalize CF by cell number (mN/cell) or cell density (mN/ cell*mm –2 ), of the muscle construct ( Xu et al, 2019 ).…”

Section: Contractile Force Analysismentioning

confidence: 99%

Contractile force assessment methods for in vitro skeletal muscle tissues

Vesga-Castro

Aldazábal

Vallejo-Illarramendi

et al. 2022

eLife

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Over the last few years, there has been growing interest in measuring the contractile force (CF) of engineered muscle tissues to evaluate their functionality. However, there are still no standards available for selecting the most suitable experimental platform, measuring system, culture protocol, or stimulation patterns. Consequently, the high variability of published data hinders any comparison between different studies. We have identified that cantilever deflection, post deflection, and force transducers are the most commonly used configurations for CF assessment in 2D and 3D models. Additionally, we have discussed the most relevant emerging technologies that would greatly complement CF evaluation with intracellular and localized analysis. This review provides a comprehensive analysis of the most significant advances in CF evaluation and its critical parameters. In order to compare contractile performance across experimental platforms, we have used the specific force (sF, kN/m2), CF normalized to the calculated cross-sectional area (CSA). However, this parameter presents a high variability throughout the different studies, which indicates the need to identify additional parameters and complementary analysis suitable for proper comparison. We propose that future contractility studies in skeletal muscle constructs report detailed information about construct size, contractile area, maturity level, sarcomere length, and, ideally, the tetanus-to-twitch ratio. These studies will hopefully shed light on the relative impact of these variables on muscle force performance of engineered muscle constructs. Prospective advances in muscle tissue engineering, particularly in muscle disease models, will require a joint effort to develop standardized methodologies for assessing CF of engineered muscle tissues.

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“…Myotubes lacking dystrophin or other members of the DGC display decreased AChR clustering ( Kong and Anderson, 1999 ), indicating that post-synaptic NMJ abnormalities can occur in the absence of neural cells. When engineered into 3D tissues, DMD primary and immortalized patient cells display decreased fusion and force of contraction, atrophic myotubes, and decreased nuclear anisotropy ( Nesmith et al, 2016 ; Al Tanoury et al, 2021 ; Ebrahimi et al, 2021 ). Large scale personalized platforms amenable to pharmacological screens will require the use of patient hiPSCs due to ethical and proliferative limitations of muscle biopsy-derived primary cells and a need for non-muscle cells such as MNs ( Wang J. et al, 2019 ).…”

Section: Neuromuscular Diseasesmentioning

confidence: 99%

Neuromuscular Development and Disease: Learning From in vitro and in vivo Models

Fralish

Lotz

Chavez

et al. 2021

Front. Cell Dev. Biol.

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The neuromuscular junction (NMJ) is a specialized cholinergic synaptic interface between a motor neuron and a skeletal muscle fiber that translates presynaptic electrical impulses into motor function. NMJ formation and maintenance require tightly regulated signaling and cellular communication among motor neurons, myogenic cells, and Schwann cells. Neuromuscular diseases (NMDs) can result in loss of NMJ function and motor input leading to paralysis or even death. Although small animal models have been instrumental in advancing our understanding of the NMJ structure and function, the complexities of studying this multi-tissue system in vivo and poor clinical outcomes of candidate therapies developed in small animal models has driven the need for in vitro models of functional human NMJ to complement animal studies. In this review, we discuss prevailing models of NMDs and highlight the current progress and ongoing challenges in developing human iPSC-derived (hiPSC) 3D cell culture models of functional NMJs. We first review in vivo development of motor neurons, skeletal muscle, Schwann cells, and the NMJ alongside current methods for directing the differentiation of relevant cell types from hiPSCs. We further compare the efficacy of modeling NMDs in animals and human cell culture systems in the context of five NMDs: amyotrophic lateral sclerosis, myasthenia gravis, Duchenne muscular dystrophy, myotonic dystrophy, and Pompe disease. Finally, we discuss further work necessary for hiPSC-derived NMJ models to function as effective personalized NMD platforms.

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De novo revertant fiber formation and therapy testing in a 3D culture model of Duchenne muscular dystrophy skeletal muscle

Cited by 33 publications

References 68 publications

Modeling Patient-Specific Muscular Dystrophy Phenotypes and Therapeutic Responses in Reprogrammed Myotubes Engineered on Micromolded Gelatin Hydrogels

Modeling Patient-Specific Muscular Dystrophy Phenotypes and Therapeutic Responses in Reprogrammed Myotubes Engineered on Micromolded Gelatin Hydrogels

Contractile force assessment methods for in vitro skeletal muscle tissues

Neuromuscular Development and Disease: Learning From in vitro and in vivo Models

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