Muscle fusogens go viral for gene delivery to skeletal muscle.

Viruses and multinucleated cells rely on fusogens to facilitate the fusion of their membranes. In this issue of Cell, Millay and colleagues demonstrate that replacing viral fusogens with mammalian skeletal muscle fusogens leads to the specific transduction of skeletal muscle and the ability to deliver gene therapy constructs in a therapeutically relevant muscle disease.

Exon Skipping Therapy.

Exondys 51 is the first therapy for Duchenne muscular dystrophy (DMD) to have been granted accelerated approval by the FDA. Approval was granted based on using dystrophin expression as a surrogate marker. Exondys 51 targets DMD exon 51 for skipping to restore the reading frame for 13% of Duchenne patients.

SIX1+PAX3+ identify a progenitor for myogenic lineage commitment from hPSCs.

The earliest skeletal muscle progenitor cells (SMPCs) derived from human pluripotent stem cells (hPSCs) are often identified by factors expressed by a diverse number of progenitors. An early transcriptional checkpoint that defines myogenic commitment could improve hPSC differentiation to skeletal muscle. Analysis of several myogenic factors in human embryos and early hPSC differentiations found SIX1+PAX3+ co-expression was most indictive of myogenesis. Using dCas9-KRAB hPSCs, we demonstrate that early inhibition of SIX1 alone significantly decreased PAX3 expression, reduced PAX7+ SMPCs, and myotubes later in differentiation. Emergence of SIX1+PAX3+ precursors can be improved by manipulating seeding density, monitoring metabolic secretion and altering the concentration of CHIR99021. These modifications resulted in the co-emergence of hPSC-derived sclerotome, cardiac and neural crest that we hypothesized enhanced hPSC myogenic differentiation. Inhibition of non-myogenic lineages modulated PAX3 independent of SIX1. To better understand SIX1 expression, we compared directed differentiations to fetal progenitors and adult satellite cells by RNA-seq. Although SIX1 continued to be expressed across human development, SIX1 co-factor expression was dependent on developmental timing. We provide a resource to enable efficient derivation of skeletal muscle from hPSCs.

Recapitulating human myogenesis ex vivo using human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) provide a human model for developmental myogenesis, disease modeling and development of therapeutics. Differentiation of hPSCs into muscle stem cells has the potential to provide a cell-based therapy for many skeletal muscle wasting diseases. This review describes the current state of hPSCs towards recapitulating human myogenesis ex vivo, considerations of stem cell and progenitor cell state as well as function for future use of hPSC-derived muscle cells in regenerative medicine.

Nanoparticles systemically biodistribute to regenerating skeletal muscle in DMD.

Skeletal muscle disease severity can often progress asymmetrically across muscle groups and heterogeneously within tissues. An example is Duchenne Muscular Dystrophy (DMD) in which lack of dystrophin results in devastating skeletal muscle wasting in some muscles whereas others are spared or undergo hypertrophy. An efficient, non-invasive approach to identify sites of asymmetry and degenerative lesions could enable better patient monitoring and therapeutic targeting of disease. In this study, we utilized a versatile intravenously injectable mesoporous silica nanoparticle (MSNP) based nanocarrier system to explore mechanisms of biodistribution in skeletal muscle of mdx mouse models of DMD including wildtype, dystrophic, and severely dystrophic mice. Moreover, MSNPs could be imaged in live mice and whole muscle tissues enabling investigation of how biodistribution is altered by different types of muscle pathology such as inflammation or fibrosis. We found MSNPs were tenfold more likely to aggregate within select mdx muscles relative to wild type, such as gastrocnemius and quadriceps. This was accompanied by decreased biodistribution in off-target organs. We found the greatest factor affecting preferential delivery was the regenerative state of the dystrophic skeletal muscle with the highest MSNP abundance coinciding with the regions showing the highest level of embryonic myosin staining and intramuscular macrophage uptake. To demonstrate, muscle regeneration regulated MSNP distribution, we experimentally induced regeneration using barium chloride which resulted in a threefold increase of intravenously injected MSNPs to sites of regeneration 7 days after injury. These discoveries provide the first evidence that nanoparticles have selective biodistribution to skeletal muscle in DMD to areas of active regeneration and that nanoparticles could enable diagnostic and selective drug delivery in DMD skeletal muscle.

CRISPR-Mediated Genomic Addition to CPS1 Deficient iPSCs is Insufficient to Restore Nitrogen Homeostasis.

CPS1 deficiency is an inborn error of metabolism caused by loss-of-function mutations in the CPS1 gene, catalyzing the initial reaction of the urea cycle. Deficiency typically leads to toxic levels of plasma ammonia, cerebral edema, coma, and death, with the only curative treatment being liver transplantation; due to limited donor availability and the invasiveness and complications of the procedure, however, alternative therapies are needed. Induced pluripotent stem cells offer an alternative cell source to partial or whole liver grafts that theoretically would not require immune suppression regimens and additionally are amenable to genetic modifications. Here, we genetically modified CPS1 deficient patient-derived stem cells to constitutively express human codon optimized CPS1 from the AAVS1 safe harbor site. While edited stem cells efficiently differentiated to hepatocyte-like cells, they failed to metabolize ammonia more efficiently than their unedited counterparts. This unexpected result appears to have arisen in part due to transgene promoter methylation, and thus transcriptional silencing, in undifferentiated cells, impacting their capacity to restore the complete urea cycle function upon differentiation. As pluripotent stem cell strategies are being expanded widely for potential cell therapies, these results highlight the need for strict quality control and functional analysis to ensure the integrity of cell products.

CRISPR for Neuromuscular Disorders: Gene Editing and Beyond.

This is a review describing advances in CRISPR/Cas-mediated therapies for neuromuscular disorders (NMDs). We explore both CRISPR-mediated editing and dead Cas approaches as potential therapeutic strategies for multiple NMDs. Last, therapeutic considerations, including delivery and off-target effects, are also discussed.

Supramolecular nanosubstrate-mediated delivery for reprogramming and transdifferentiation of mammalian cells.

Supramolecular nanosubstrate-mediated delivery (SNSMD) leverages the power of molecular self-assembly and a nanostructured substrate platform for the low toxicity, highly efficient co-delivery of biological factors encapsulated in a nanovector. Human fibroblasts are successfully reprogrammed into induced pluripotent stems and transdifferentiated into induced neuronal-like cells.

Nipah virus envelope-pseudotyped lentiviruses efficiently target ephrinB2-positive stem cell populations in vitro and bypass the liver sink when administered in vivo.

Sophisticated retargeting systems for lentiviral vectors have been developed in recent years. Most seek to suppress the viral envelope's natural tropism while modifying the receptor-binding domain such that its tropism is determined by the specificity of the engineered ligand-binding motif. Here we took advantage of the natural tropism of Nipah virus (NiV), whose attachment envelope glycoprotein has picomolar affinity for ephrinB2, a molecule proposed as a molecular marker of "stemness" (present on embryonic, hematopoietic, and neural stem cells) as well as being implicated in tumorigenesis of specific cancers. NiV entry requires both the fusion (F) and attachment (G) glycoproteins. Truncation of the NiV-F cytoplasmic tail (T5F) alone, combined with full-length NiV-G, resulted in optimal titers of NiV-pseudotyped particles (NiVpp) (∼10(6) IU/ml), even without ultracentrifugation. To further enhance the infectivity of NiVpp, we engineered a hyperfusogenic NiV-F protein lacking an N-linked glycosylation site (T5FΔN3). T5FΔN3/wt G particles exhibited enhanced infectivity on less permissive cell lines and efficiently targeted ephrinB2(+) cells even in a 1,000-fold excess of ephrinB2-negative cells, all without any loss of specificity, as entry was abrogated by soluble ephrinB2. NiVpp also transduced human embryonic, hematopoietic, and neural stem cell populations in an ephrinB2-dependent manner. Finally, intravenous administration of the luciferase reporter NiVpp-T5FΔN3/G to mice resulted in signals being detected in the spleen and lung but not in the liver. Bypassing the liver sink is a critical barrier for targeted gene therapy. The extraordinary specificity of NiV-G for ephrinB2 holds promise for targeting specific ephrinB2(+) populations in vivo or in vitro.

Involvement of lysosomal exocytosis in the excretion of mesoporous silica nanoparticles and enhancement of the drug delivery effect by exocytosis inhibition.

The exocytosis of phosphonate modified mesoporous silica nanoparticles (P-MSNs) is demonstrated and lysosomal exocytosis is identified as the mechanism responsible for this event. Regulation of P-MSN exocytosis can be achieved by inhibiting or accelerating lysosomal exocytosis. Slowing down P-MSN exocytosis enhances the drug delivery effect of CPT-loaded P-MSNs by improving cell killing.

The emergence of the stem cell niche.

Stem cell niches are composed of dynamic microenvironments that support stem cells over a lifetime. The emerging niche is distinct from the adult because its main role is to support the progenitors that build organ systems in development. Emerging niches mature through distinct stages to form the adult niche and enable proper stem cell support. As a model of emerging niches, this review highlights how differences in the skeletal muscle microenvironment influence emerging versus satellite cell (SC) niche formation in skeletal muscle, which is among the most regenerative tissue systems. We contrast how stem cell niches regulate intrinsic properties between progenitor and stem cells throughout development to adulthood. We describe new applications for generating emerging niches from human pluripotent stem cells (hPSCs) using developmental principles and highlight potential applications for regeneration and therapeutics.

'Enhancing' skeletal muscle and stem cells in three-dimensions: genome regulation of skeletal muscle in development and disease.

The noncoding genome imparts important regulatory control over gene expression. In particular, gene enhancers represent a critical layer of control that integrates developmental and differentiation signals outside the cell into transcriptional outputs inside the cell. Recently, there has been an explosion in genomic techniques to probe enhancer control, function, and regulation. How enhancers are regulated and integrate signals in stem cell development and differentiation is largely an open question. In this review, we focus on the role gene enhancers play in muscle stem cell specification, differentiation, and progression. We pay specific attention toward the identification of muscle-specific enhancers, the binding of transcription factors to these enhancers, and how enhancers communicate to their target genes via three-dimensional looping.

Duchenne muscular dystrophy disease severity impacts skeletal muscle progenitor cells systemic delivery.

Duchenne muscular dystrophy (DMD) is caused by an out-of-frame mutation in the DMD gene that results in the absence of a functional dystrophin protein, leading to a devastating progressive lethal muscle-wasting disease. Muscle stem cell-based therapy is a promising avenue for improving muscle regeneration. However, despite the efforts to deliver the optimal cell population to multiple muscles most efforts have failed. Here we describe a detailed optimized method of for the delivery of human skeletal muscle progenitor cells (SMPCs) to multiple hindlimb muscles in healthy, dystrophic and severely dystrophic mouse models. We show that systemic delivery is inefficient and is affected by the microenvironment. We found that significantly less human SMPCs were detected in healthy gastrocnemius muscle cross-sections, compared to both dystrophic and severely dystrophic gastrocnemius muscle. Human SMPCs were found to be detected inside blood vessels distinctly in healthy, dystrophic and severely dystrophic muscles, with prominent clotting identified in severely dystrophic muscles after intra arterial (IA) systemic cell delivery. We propose that muscle microenvironment and the severity of muscular dystrophy to an extent impacts the systemic delivery of SMPCs and that overall systemic stem cell delivery is not currently efficient or safe to be used in cell based therapies for DMD. This work extends our understanding of the severe nature of DMD, which should be taken into account when considering stem cell-based systemic delivery platforms.

Regenerating human skeletal muscle forms an emerging niche in vivo to support PAX7 cells.

Skeletal muscle stem and progenitor cells including those derived from human pluripotent stem cells (hPSCs) offer an avenue towards personalized therapies and readily fuse to form human-mouse myofibres in vivo. However, skeletal muscle progenitor cells (SMPCs) inefficiently colonize chimeric stem cell niches and instead associate with human myofibres resembling foetal niches. We hypothesized competition with mouse satellite cells (SCs) prevented SMPC engraftment into the SC niche and thus generated an SC ablation mouse compatible with human engraftment. Single-nucleus RNA sequencing of SC-ablated mice identified the absence of a transient myofibre subtype during regeneration expressing Actc1. Similarly, ACTC1+ human myofibres supporting PAX7+ SMPCs increased in SC-ablated mice, and after re-injury we found SMPCs could now repopulate into chimeric niches. To demonstrate ACTC1+ myofibres are essential to supporting PAX7 SMPCs, we generated caspase-inducible ACTC1 depletion human pluripotent stem cells, and upon SMPC engraftment we found a 90% reduction in ACTC1+ myofibres and a 100-fold decrease in PAX7 cell numbers compared with non-induced controls. We used spatial RNA sequencing to identify key factors driving emerging human niche formation between ACTC1+ myofibres and PAX7+ SMPCs in vivo. This revealed that transient regenerating human myofibres are essential for emerging niche formation in vivo to support PAX7 SMPCs.

Innate and adaptive AAV-mediated immune responses in a mouse model of Duchenne muscular dystrophy.

High systemic doses of adeno-associated viruses (AAVs) have been associated with immune-related serious adverse events (SAEs). Although AAV was well tolerated in preclinical models, SAEs were observed in clinical trials, indicating the need for improved preclinical models to understand AAV-induced immune responses. Here, we show that mice dual-dosed with AAV9 at 4-week intervals better recapitulate aspects of human immunity to AAV. In the model, anti-AAV9 immunoglobulin G (IgGs) increased in a linear fashion between the first and second AAV administrations. Complement activation was only observed in the presence of high levels of both AAV and anti-AAV IgG. Myeloid-derived pro-inflammatory cytokines were significantly induced in the same pattern as complement activation, suggesting that myeloid cell activation to AAV may rely on the presence of both AAV and anti-AAV IgG complexes. Single-cell RNA sequencing of peripheral blood mononuclear cells confirmed that activated monocytes were a primary source of pro-inflammatory cytokines and chemokines, which were significantly increased after a second AAV9 exposure. The same activated monocyte clusters expressed both Fcγ and complement receptors, suggesting that anti-AAV-mediated activation of myeloid cells through Fcγ receptors and/or complement receptors is one mechanism by which anti-AAV antigen complexes may prime antigen-presenting cells and amplify downstream immunity.

Myoscaffolds reveal laminin scarring is detrimental for stem cell function while sarcospan induces compensatory fibrosis.

We developed an on-slide decellularization approach to generate acellular extracellular matrix (ECM) myoscaffolds that can be repopulated with various cell types to interrogate cell-ECM interactions. Using this platform, we investigated whether fibrotic ECM scarring affected human skeletal muscle progenitor cell (SMPC) functions that are essential for myoregeneration. SMPCs exhibited robust adhesion, motility, and differentiation on healthy muscle-derived myoscaffolds. All SPMC interactions with fibrotic myoscaffolds from dystrophic muscle were severely blunted including reduced motility rate and migration. Furthermore, SMPCs were unable to remodel laminin dense fibrotic scars within diseased myoscaffolds. Proteomics and structural analysis revealed that excessive collagen deposition alone is not pathological, and can be compensatory, as revealed by overexpression of sarcospan and its associated ECM receptors in dystrophic muscle. Our in vivo data also supported that ECM remodeling is important for SMPC engraftment and that fibrotic scars may represent one barrier to efficient cell therapy.

Phosphatase and tensin homolog regulates the pluripotent state and lineage fate choice in human embryonic stem cells.

Understanding the intrinsic and extrinsic signals that regulate the molecular basis of the pluripotent state may improve our understanding of mammalian embryogenesis, different states of pluripotency, and our ability to tailor lineage differentiation. Although the role of the PI3K/Akt pathway in the self-renewal and maintenance of mESCs is well-established, the specific contribution of the pathway or of its negative regulator, PTEN, in the maintenance of the human pluripotent state is less understood. To explore the PI3K/AKT pathway in human embryonic stem cell (hESC) pluripotency and differentiation, we generated stable PTEN knockdown (KD) hESCs using short hairpin RNA. Similar to mESCs, we found that PTEN KD hESCs have increased self-renewal, cell survival, and proliferation over multiple passages compared to control cells. However, in contrast to mESCs, in vitro, PTEN KD hESCs differentiated inefficiently in directed differentiation assays, in part due to the continued maintenance of OCT4 and NANOG expression. In teratoma assays, PTEN KD hESCs generated tissues from the three germ layers, although with a bias toward neuroectoderm differentiation. These results demonstrate that PTEN is a key regulator of hESC growth and differentiation, and manipulation of this pathway may improve our ability to regulate and understand the pluripotent state.

Single cell sequencing maps skeletal muscle cellular diversity as disease severity increases in dystrophic mouse models.

Duchenne muscular dystrophy (DMD) is caused by out-of-frame mutations in the DMD gene resulting in the absence of a functional dystrophin protein, leading to a devastating and progressive lethal muscle-wasting disease. Little is known about cellular heterogeneity as disease severity increases. Advances in single-cell RNA sequencing (scRNA-seq) enabled us to explore skeletal muscle-resident cell populations in healthy, dystrophic, and severely dystrophic mouse models. We found increased frequencies of activated fibroblasts, fibro-adipogenic progenitor cells, and pro-inflammatory macrophages in dystrophic gastrocnemius muscles and an upregulation of extracellular matrix genes on endothelial cells in dystrophic and severely dystrophic muscles. We observed a pronounced risk of clotting, especially in the severely dystrophic mice with increased expression of plasminogen activator inhibitor-1 in endothelial cells, indicating endothelial cell impairment as disease severity increases. This work extends our understanding of the severe nature of DMD which should be considered when developing single or combinatorial approaches for DMD.

Single-cell analysis and functional characterization uncover the stem cell hierarchies and developmental origins of rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a common childhood cancer that shares features with developing skeletal muscle. Yet, the conservation of cellular hierarchy with human muscle development and the identification of molecularly defined tumor-propagating cells has not been reported. Using single-cell RNA-sequencing, DNA-barcode cell fate mapping and functional stem cell assays, we uncovered shared tumor cell hierarchies in RMS and human muscle development. We also identified common developmental stages at which tumor cells become arrested. Fusion-negative RMS cells resemble early myogenic cells found in embryonic and fetal development, while fusion-positive RMS cells express a highly specific gene program found in muscle cells transiting from embryonic to fetal development at 7-7.75 weeks of age. Fusion-positive RMS cells also have neural pathway-enriched states, suggesting less-rigid adherence to muscle-lineage hierarchies. Finally, we identified a molecularly defined tumor-propagating subpopulation in fusion-negative RMS that shares remarkable similarity to bi-potent, muscle mesenchyme progenitors that can make both muscle and osteogenic cells.

Long-term repair of porcine articular cartilage using cryopreservable, clinically compatible human embryonic stem cell-derived chondrocytes.

Osteoarthritis (OA) impacts hundreds of millions of people worldwide, with those affected incurring significant physical and financial burdens. Injuries such as focal defects to the articular surface are a major contributing risk factor for the development of OA. Current cartilage repair strategies are moderately effective at reducing pain but often replace damaged tissue with biomechanically inferior fibrocartilage. Here we describe the development, transcriptomic ontogenetic characterization and quality assessment at the single cell level, as well as the scaled manufacturing of an allogeneic human pluripotent stem cell-derived articular chondrocyte formulation that exhibits long-term functional repair of porcine articular cartilage. These results define a new potential clinical paradigm for articular cartilage repair and mitigation of the associated risk of OA.