Seeding competent TDP-43 persists in human patient and mouse muscle.

TAR DNA-binding protein 43 (TDP-43) is an RNA binding protein that accumulates as aggregates in the central nervous system of some neurodegenerative diseases. However, TDP-43 aggregation is also a sensitive and specific pathologic feature found in a family of degenerative muscle diseases termed inclusion body myopathy (IBM). TDP-43 aggregates from ALS and FTD brain lysates may serve as self-templating aggregate seeds in vitro and in vivo, supporting a prion-like spread from cell to cell. Whether a similar process occurs in IBM patient muscle is not clear. We developed a mouse model of inducible, muscle-specific cytoplasmic localized TDP-43. These mice develop muscle weakness with robust accumulation of insoluble and phosphorylated sarcoplasmic TDP-43, leading to eosinophilic inclusions, altered proteostasis and changes in TDP-43-related RNA processing that resolve with the removal of doxycycline. Skeletal muscle lysates from these mice also have seeding competent TDP-43, as determined by a FRET-based biosensor, that persists for weeks upon resolution of TDP-43 aggregate pathology. Human muscle biopsies with TDP-43 pathology also contain TDP-43 aggregate seeds. Using lysates from muscle biopsies of patients with IBM, IMNM and ALS we found that TDP-43 seeding capacity was specific to IBM. Surprisingly, TDP-43 seeding capacity anti-correlated with TDP-43 aggregate and vacuole abundance. These data support that TDP-43 aggregate seeds are present in IBM skeletal muscle and represent a unique TDP-43 pathogenic species not previously appreciated in human muscle disease.

Current Progress in the Creation, Characterization, and Application of Human Stem Cell-derived in Vitro Neuromuscular Junction Models.

Human pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) are of great value for studying developmental processes, disease modeling, and drug testing. One area in which the use of human PSCs has become of great interest in recent years is for in vitro models of the neuromuscular junction (NMJ). The NMJ is a synapse at which a motor neuron releases acetylcholine to bind to skeletal muscle and stimulate contraction. Degeneration of the NMJ and subsequent loss of muscle function is a common feature of many neuromuscular diseases such as myasthenia gravis, spinal muscular atrophy, and amyotrophic lateral sclerosis. In order to develop new therapies for patients with neuromuscular diseases, it is essential to understand mechanisms taking place at the NMJ. However, we have limited ability to study the NMJ in living human patients, and animal models are limited by physiological relevance. Therefore, an in vitro model of the NMJ consisting of human cells is of great value. The use of stem cells for in vitro NMJ models is still in progress and requires further optimization in order to yield reliable, reproducible results. The objective of this review is (1) to outline the current progress towards fully PSC-derived in vitro co-culture models of the human NMJ and (2) to discuss future directions and challenges that must be overcome in order to create reproducible fully PSC-derived models that can be used for developmental studies, disease modeling, and drug testing.

Transcriptome analysis using patient iPSC-derived skeletal myocytes: Bet1L as a new molecule possibly linked to neuromuscular junction degeneration in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease in which patients gradually become paralyzed due to loss of motor function. Many genetically inheritable mutations have been linked to ALS; however, the majority of ALS patients are considered sporadic. Therefore, there is a need for a common therapy that is effective for all ALS patients. Although there is evidence of the disease beginning in the periphery at the neuromuscular junction (NMJ), the specific processes involved in skeletal muscle and at the NMJ are still largely unknown. To study common disease mechanisms in ALS skeletal muscle, we performed RNA sequencing of skeletal myocytes differentiated from induced pluripotent stem cells (iPSCs) derived from familial ALS (with C9ORF72, SOD1, or TARDBP mutations) and sporadic ALS patients. Compared to healthy control lines, the myocytes from all ALS lines showed downregulation of four genes: BET1L, DCX, GPC3, and HNRNPK. We next measured the expression levels of these four genes in hind limb muscle samples from a rat model of familial ALS (SOD1G93A transgenic) and found that only the Bet1L gene, which encodes Bet1 Golgi Vesicular Membrane Trafficking Protein Like, was commonly downregulated. Bet1L protein appeared to be localized to the basal lamina of the NMJ, with decreased expression over time in SOD1G93A transgenic rats. Importantly, the expression levels began to decrease early in the disease process. Our results indicate that loss of Bet1L at the NMJ could be of interest for better understanding ALS disease progression.

Coding Cell Identity of Human Skeletal Muscle Progenitor Cells Using Cell Surface Markers: Current Status and Remaining Challenges for Characterization and Isolation.

Skeletal muscle progenitor cells (SMPCs), also called myogenic progenitors, have been studied extensively in recent years because of their promising therapeutic potential to preserve and recover skeletal muscle mass and function in patients with cachexia, sarcopenia, and neuromuscular diseases. SMPCs can be utilized to investigate the mechanisms of natural and pathological myogenesis via in vitro modeling and in vivo experimentation. While various types of SMPCs are currently available from several sources, human pluripotent stem cells (PSCs) offer an efficient and cost-effective method to derive SMPCs. As human PSC-derived cells often display varying heterogeneity in cell types, cell enrichment using cell surface markers remains a critical step in current procedures to establish a pure population of SMPCs. Here we summarize the cell surface markers currently being used to detect human SMPCs, describing their potential application for characterizing, identifying and isolating human PSC-derived SMPCs. To date, several positive and negative markers have been used to enrich human SMPCs from differentiated PSCs by cell sorting. A careful analysis of current findings can broaden our understanding and reveal potential uses for these surface markers with SMPCs.

C9ORF72-related cellular pathology in skeletal myocytes derived from ALS-patient induced pluripotent stem cells.

Amyotrophic lateral sclerosis (ALS) is a late-onset neuromuscular disease with no cure and limited treatment options. Patients experience a gradual paralysis leading to death from respiratory complications on average only 2-5 years after diagnosis. There is increasing evidence that skeletal muscle is affected early in the disease process, yet the pathological processes occurring in the skeletal muscle of ALS patients are still mostly unknown. Specifically, the most common genetic cause of ALS, a hexanucleotide repeat expansion in the C9ORF72 gene, has yet to be fully characterized in the context of skeletal muscle. In this study, we used the protocol previously developed in our lab to differentiate skeletal myocytes from induced pluripotent stem cells (iPSCs) of C9ORF72 ALS (C9-ALS) patients in order to create an in vitro disease model of C9-ALS skeletal muscle pathology. Of the three C9ORF72 mutation hallmarks, we did not see any evidence of haploinsufficiency, but we did detect RNA foci and dipeptide repeat (DPR) proteins. Additional abnormalities included changes in the expression of mitochondrial genes and a susceptibility to oxidative stress, indicating that mitochondrial dysfunction may be a critical feature of C9-ALS skeletal muscle pathology. Finally, the C9-ALS myocytes had increased expression and aggregation of TDP-43. Together, these data show that skeletal muscle cells experience pathological changes due to the C9ORF72 mutation. Our in vitro model could facilitate further study of cellular and molecular pathology in ALS skeletal muscle in order to discover new therapeutic targets against this devastating disease.This article has an associated First Person interview with the first author of the paper.

Micropatterned substrates with physiological stiffness promote cell maturation and Pompe disease phenotype in human induced pluripotent stem cell-derived skeletal myocytes.

Recent advances in bioengineering have enabled cell culture systems that more closely mimic the native cellular environment. Here, we demonstrated that human induced pluripotent stem cell (iPSC)-derived myogenic progenitors formed highly-aligned myotubes and contracted when seeded on two-dimensional micropatterned platforms. The differentiated cells showed clear nuclear alignment and formed elongated myotubes dependent on the width of the micropatterned lanes. Topographical cues from micropatterning and physiological substrate stiffness improved the formation of well-aligned and multinucleated myotubes similar to myofibers. These aligned myotubes exhibited spontaneous contractions specifically along the long axis of the pattern. Notably, the micropatterned platforms developed bundle-like myotubes using patient-derived iPSCs with a background of Pompe disease (glycogen storage disease type II) and even enhanced the disease phenotype as shown through the specific pathology of abnormal lysosome accumulations. A highly-aligned formation of matured myotubes holds great potential in further understanding the process of human muscle development, as well as advancing in vitro pharmacological studies for skeletal muscle diseases.

Current Progress and Challenges for Skeletal Muscle Differentiation from Human Pluripotent Stem Cells Using Transgene-Free Approaches.

Neuromuscular diseases are caused by functional defects of skeletal muscles, directly via muscle pathology or indirectly via disruption of the nervous system. Extensive studies have been performed to improve the outcomes of therapies; however, effective treatment strategies have not been fully established for any major neuromuscular disease. Human pluripotent stem cells have a great capacity to differentiate into myogenic progenitors and skeletal myocytes for use in treating and modeling neuromuscular diseases. Recent advances have allowed the creation of patient-derived stem cells, which can be used as a unique platform for comprehensive study of disease mechanisms, in vitro drug screening, and potential new cell-based therapies. In the last decade, a number of methods have been developed to derive skeletal muscle cells from human pluripotent stem cells. By controlling the process of myogenesis using transcription factors and signaling molecules, human pluripotent stem cells can be directed to differentiate into cell types observed during muscle development. In this review, we highlight signaling pathways relevant to the formation of muscle tissue during embryonic development. We then summarize current methods to differentiate human pluripotent stem cells toward the myogenic lineage, specifically focusing on transgene-free approaches. Lastly, we discuss existing challenges for deriving skeletal myocytes and myogenic progenitors from human pluripotent stem cells.

Differentiation and sarcomere formation in skeletal myocytes directly prepared from human induced pluripotent stem cells using a sphere-based culture.

Human induced-pluripotent stem cells (iPSCs) are a promising resource for propagation of myogenic progenitors. Our group recently reported a unique protocol for the derivation of myogenic progenitors directly (without genetic modification) from human pluripotent cells using free-floating spherical culture. Here we expand our previous efforts and attempt to determine how differentiation duration, culture surface coatings, and nutrient supplements in the medium influence progenitor differentiation and formation of skeletal myotubes containing sarcomeric structures. A long differentiation period (over 6 weeks) promoted the differentiation of iPSC-derived myogenic progenitors and subsequent myotube formation. These iPSC-derived myotubes contained representative sarcomeric structures, consisting of organized myosin and actin filaments, and could spontaneously contract. We also found that a bioengineering approach using three-dimensional (3D) artificial muscle constructs could facilitate the formation of elongated myotubes. Lastly, we determined how culture surface coating matrices and different supplements would influence terminal differentiation. While both Matrigel and laminin coatings showed comparable effects on muscle differentiation, B27 serum-free supplement in the differentiation medium significantly enhanced myogenesis compared to horse serum. Our findings support the possibility to create an in vitro model of contractile sarcomeric myofibrils for disease modeling and drug screening to study neuromuscular diseases.