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.

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.

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.

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.

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.

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.

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.

A customizable microfluidic platform for medium-throughput modeling of neuromuscular circuits.

Neuromuscular circuits (NMCs) are vital for voluntary movement, and effective models of NMCs are needed to understand the pathogenesis of, as well as to identify effective treatments for, multiple diseases, including Duchenne's muscular dystrophy and amyotrophic lateral sclerosis. Microfluidics are ideal for recapitulating the central and peripheral compartments of NMCs, but myotubes often detach before functional NMCs are formed. In addition, microfluidic systems are often limited to a single experimental unit, which significantly limits their application in disease modeling and drug discovery. Here, we developed a microfluidic platform (MFP) containing over 100 experimental units, making it suitable for medium-throughput applications. To overcome detachment, we incorporated a reactive polymer surface allowing customization of the environment to culture different cell types. Using this approach, we identified conditions that enable long-term co-culture of human motor neurons and myotubes differentiated from human induced pluripotent stem cells inside our MFP. Optogenetics demonstrated the formation of functional NMCs. Furthermore, we developed a novel application of the rabies tracing assay to efficiently identify NMCs in our MFP. Therefore, our MFP enables large-scale generation and quantification of functional NMCs for disease modeling and pharmacological drug targeting.

ERBB3 and NGFR mark a distinct skeletal muscle progenitor cell in human development and hPSCs.

Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progenitor cells (SMPCs). However, the myogenicity of hPSC-SMPCs relative to human fetal or adult satellite cells remains unclear. We observed that hPSC-SMPCs derived by directed differentiation are less functional in vitro and in vivo compared to human satellite cells. Using RNA sequencing, we found that the cell surface receptors ERBB3 and NGFR demarcate myogenic populations, including PAX7 progenitors in human fetal development and hPSC-SMPCs. We demonstrated that hPSC skeletal muscle is immature, but inhibition of transforming growth factor-ß signalling during differentiation improved fusion efficiency, ultrastructural organization and the expression of adult myosins. This enrichment and maturation strategy restored dystrophin in hundreds of dystrophin-deficient myofibres after engraftment of CRISPR-Cas9-corrected Duchenne muscular dystrophy human induced pluripotent stem cell-SMPCs. The work provides an in-depth characterization of human myogenesis, and identifies candidates that improve the in vivo myogenic potential of hPSC-SMPCs to levels that are equal to directly isolated human fetal muscle cells.

A Single CRISPR-Cas9 Deletion Strategy that Targets the Majority of DMD Patients Restores Dystrophin Function in hiPSC-Derived Muscle Cells.

Mutations in DMD disrupt the reading frame, prevent dystrophin translation, and cause Duchenne muscular dystrophy (DMD). Here we describe a CRISPR/Cas9 platform applicable to 60% of DMD patient mutations. We applied the platform to DMD-derived hiPSCs where successful deletion and non-homologous end joining of up to 725 kb reframed the DMD gene. This is the largest CRISPR/Cas9-mediated deletion shown to date in DMD. Use of hiPSCs allowed evaluation of dystrophin in disease-relevant cell types. Cardiomyocytes and skeletal muscle myotubes derived from reframed hiPSC clonal lines had restored dystrophin protein. The internally deleted dystrophin was functional as demonstrated by improved membrane integrity and restoration of the dystrophin glycoprotein complex in vitro and in vivo. Furthermore, miR31 was reduced upon reframing, similar to observations in Becker muscular dystrophy. This work demonstrates the feasibility of using a single CRISPR pair to correct the reading frame for the majority of DMD patients.

Duration and magnitude of myofascial release in 3-dimensional bioengineered tendons: effects on wound healing.

CONTEXT: Myofascial release (MFR) is one of the most commonly used manual manipulative treatments for patients with soft tissue injury. However, a paucity of basic science evidence has been published to support any particular mechanism that may contribute to reported clinical efficacies of MFR. OBJECTIVE: To investigate the effects of duration and magnitude of MFR strain on wound healing in bioengineered tendons (BETs) in vitro. METHODS: The BETs were cultured on a deformable matrix and then wounded with a steel cutting tip. Using vacuum pressure, they were then strained with a modeled MFR paradigm. The duration of MFR dose consisted of a slow-loading strain that stretched the BETs 6% beyond their resting length, held them for 0, 1, 2, 3, 4, or 5 minutes, and then slowly released them back to baseline. To assess the effects of MFR magnitude, the BETs were stretched to 0%, 3%, 6%, 9%, or 12% beyond resting length, held for 90 seconds, and then released back to baseline. Repeated measures of BET width and the wound's area, shape, and major and minor axes were quantified using microscopy over a 48-hour period. RESULTS: An 11% and 12% reduction in BET width were observed in groups with a 9% (0.961 mm; P<.01) and 12% (0.952 mm; P<.05) strain, respectively. Reduction of the minor axis of the wound was unrelated to changes in BET width. In the 3% strain group, a statistically significant decrease (-40%; P<.05) in wound size was observed at 24 hours compared with 48 hours in the nonstrain, 6% strain, and 9% strain groups. Longer duration of MFR resulted in rapid decreases in wound size, which were observed as early as 3 hours after strain. CONCLUSION: Wound healing is highly dependent on the duration and magnitude of MFR strain, with a lower magnitude and longer duration leading to the most improvement. The rapid change in wound area observed 3 hours after strain suggests that this phenomenon is likely a result of the modification of the existing matrix protein architecture. These data suggest that MFR's effect on the extracellular matrix can potentially promote wound healing.

Biomechanical strain vehicles for fibroblast-directed skeletal myoblast differentiation and myotube functionality in a novel coculture.

Skeletal muscle functionality is governed by multiple stimuli, including cytokines and biomechanical strain. Fibroblasts embedded within muscle connective tissue respond to biomechanical strain by secreting cytokines that induce myoblast differentiation and, we hypothesize, regulate myotube function. A coculture was established to allow cross talk between fibroblasts in Bioflex wells and myoblasts on nondeformable coverslips situated above Bioflex wells. Cyclic short-duration strain (CSDS) modeling repetitive stress/injury, acyclic long-duration strain (ALDS) modeling manipulative therapy, and combined strain paradigms (CSDS + ALDS) were applied to fibroblasts. Nonstrained myoblasts in uniculture and coculture served as controls. After fibroblasts had induced myoblast differentiation, myotube contraction was assessed by perfusion of ACh (10(-11)-10(-3) M). CSDS-treated fibroblasts increased myotube contractile sensitivity vs. uniculture (P < 0.05). As contraction is dependent on ACh binding, expression and clustering of nicotinic ACh receptors (nAChRs) were measured. CSDS-treated fibroblasts increased nAChR expression (P < 0.05), which correlated with myotube contraction. ALDS-treated fibroblasts did not significantly affect contraction or nAChR expression. Agrin-treated myotubes were then used to design a computer algorithm to identify α-bungarotoxin-stained nAChR clusters. ALDS-treated fibroblasts increased nAChR clustering (P < 0.05), while CSDS-treated fibroblasts disrupted cluster formation. CSDS-treated fibroblasts produced nAChRs preferentially located in nonclustered regions (P < 0.05). Strain-activated fibroblasts mediate myotube differentiation with multiple functional phenotypes. Similar to muscle injury, CSDS-treated fibroblasts disrupted nAChR clusters and hypersensitized myotube contraction, while ALDS-treated fibroblasts aggregated nAChRs in large clusters, which may have important clinical implications. Cellular strategies aimed at improving muscle functionality, such as through biomechanical strain vehicles that activate fibroblasts to stabilize postsynaptic nAChRs on nearby skeletal muscle, may serve as novel targets in neuromuscular disorders.

In vitro biomechanical strain regulation of fibroblast wound healing.

CONTEXT: Strain-directed therapy such as vacuum compression and manual manipulative therapies are clinically effective, but their cellular and molecular mechanisms are not well understood. OBJECTIVE: To determine the effects of modeled myofascial release (MFR) on fibroblast wound healing and to investigate the potential role of nitric oxide (NO) in mediating these responses. METHODS: Using an in vitro scratch wound strain model, the authors investigated human fibroblast wound healing characteristics in response to injurious repetitive motion strain (RMS) and MFR. Secretion of NO was induced with interleukin-1ß and sodium nitroprusside and inhibited with NO synthase inhibitor L-N(G)-monomethyl arginine citrate (L-NMMA) to determine the effects of NO on wound healing. Protein microarray was also performed to evaluate the expression of intracellular protein and activation of protein kinase G (PKG), extracellular signal-regulated kinase (ERK1/2), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K), the downstream effectors in the NO pathway. RESULTS: Fibroblasts that received RMS resulted in reduced wound closure rates (vs nonstrain, P<.05), which are partially attenuated by a single dose of MFR. Interleukin-1ß and exogenous NO did not appear to have an effect on nonstrained fibroblast wound healing. However, strained fibroblasts appeared to express increased sensitivity to NO. The authors also observed a 12.2% increase in NO secretion, an increase in PKG activation, and a downregulation of PKC and PI3K inhibitory domain in the combined strain group. CONCLUSION: If clinically translatable, these data suggest that mechanical strain such as vacuum compression therapy and manual manipulative therapy may modify PKC and PI3K to sensitize fibroblasts to NO and improve wound healing by promoting cell proliferation and migration by means of PKC and PKG signaling.

Dosed myofascial release in three-dimensional bioengineered tendons: effects on human fibroblast hyperplasia, hypertrophy, and cytokine secretion.

OBJECTIVE: The purpose of this study was to investigate potential differences of magnitudes and durations associated with dosed myofascial release (MFR) on human fibroblast proliferation, hypertrophy, and cytokine secretions. METHODS: Bioengineered tendons (BETs) attached to nylon mesh anchors were strained uniaxially using a vacuum pressure designed to model MFR varying in magnitudes (0%, 3%, 6%, 9%, and 12% elongation) and durations (0.5 and 1-5 minutes). Conditioned media were analyzed for cytokine secretion via protein microarray (n = 2). Bioengineered tendons were weighted and fibroblasts extracted from the BET were assessed for total cell protein and proliferation via double-stranded DNA quantification (n = 5). All data were compared by a 1-way analysis of variance with post hoc Dunnett test and Student t test. RESULTS: Changing MFR magnitude and duration did not have an effect on total fibroblast cellular protein or DNA accumulation. However, we observed a stepwise increase in BET weight with higher-magnitude MFR treatments. Longer durations of MFR resulted in progressive increase in the secretions of angiogenin, interleukin (IL)-3, IL-8, growth colony-stimulating factor, and thymus activation-regulated chemokine. Alternatively, increasing strain magnitude induced secretions of IL-1ß, monocyte chemoattractant cytokine, and regulated and normal T cell expressed and secreted chemotactic cytokine. CONCLUSION: Cellular proliferation and hypertrophy were not significantly changed by any treatment. However, the change in total BET dry weight suggests that production of extracellular matrix protein may be up-regulated. Different MFR parameters induce secretions of a unique subset of cytokines and growth factors that can be further enhanced by increasing the magnitude and duration of treatment. If clinically translatable, these results suggest that variations to manual therapy biomechanical parameters may differentially affect physiological responses in vivo.

Mechanical strain applied to human fibroblasts differentially regulates skeletal myoblast differentiation.

Cyclic short-duration stretches (CSDS) such as those resulting from repetitive motion strain increase the risk of musculoskeletal injury. Myofascial release is a common technique used by clinicians that applies an acyclic long-duration stretch (ALDS) to muscle fascia to repair injury. When subjected to mechanical strain, fibroblasts within muscle fascia secrete IL-6, which has been shown to induce myoblast differentiation, essential for muscle repair. We hypothesize that fibroblasts subjected to ALDS following CSDS induce myoblast differentiation through IL-6. Fibroblast conditioned media and fibroblast-myoblast cocultures were used to test fibroblasts' ability to induce myoblast differentiation. The coculture system applies strain to fibroblasts only but still allows for diffusion of potential differentiation mediators to unstrained myoblasts on coverslips. To determine the role of IL-6, we utilized myoblast unicultures ± IL-6 (0-100 ng/ml) and cocultures ± α-IL-6 (0-200 µg/ml). Untreated uniculture myoblasts served as a negative control. After 96 h, coverslips (n = 6-21) were microscopically analyzed and quantified by blinded observer for differentiation endpoints: myotubes per square millimeter (>3 nuclei/cell), nuclei/myotube, and fusion efficiency (%nuclei within myotubes). The presence of fibroblasts and fibroblast conditioned media significantly enhanced myotube number (P < 0.05). However, in coculture, CSDS applied to fibroblasts did not reproduce this effect. ALDS following CSDS increased myotube number by 78% and fusion efficiency by 96% vs. CSDS alone (P < 0.05). Fibroblasts in coculture increase IL-6 secretion; however, IL-6 secretion did not correlate with enhanced differentiation among strain groups. Exogenous IL-6 in myoblast uniculture failed to induce differentiation. However, α-IL-6 attenuated differentiation in all coculture groups (P < 0.05). Fibroblasts secrete soluble mediators that have profound effects on several measures of myoblast differentiation. Specific biophysical strain patterns modify these outcomes, and suggest that myofascial release after repetitive strain increases myoblast differentiation and thus may improve muscle repair in vivo. Neutralization of IL-6 in coculture significantly reduced differentiation, suggesting fibroblast-IL-6 is necessary but not sufficient in this process.

Cyclic strain upregulates VEGF and attenuates proliferation of vascular smooth muscle cells.

OBJECTIVE: Vascular smooth muscle cell (VSMC) hypertrophy and proliferation occur in response to strain-induced local and systemic inflammatory cytokines and growth factors which may contribute to hypertension, atherosclerosis, and restenosis. We hypothesize VSMC strain, modeling normotensive arterial pressure waveforms in vitro, results in attenuated proliferative and increased hypertrophic responses 48 hrs post-strain. METHODS: Using Flexcell Bioflex Systems we determined the morphological, hyperplastic and hypertrophic responses of non-strained and biomechanically strained cultured rat A7R5 VSMC. We measured secretion of nitric oxide, key cytokine/growth factors and intracellular mediators involved in VSMC proliferation via fluorescence spectroscopy and protein microarrays. We also investigated the potential roles of VEGF on VSMC strain-induced proliferation. RESULTS: Protein microarrays revealed significant increases in VEGF secretion in response to 18 hours mechanical strain, a result that ELISA data corroborated. Apoptosis-inducing nitric oxide (NO) levels also increased 43% 48 hrs post-strain. Non-strained cells incubated with exogenous VEGF did not reproduce the antimitogenic effect. However, anti-VEGF reversed the antimitogenic effect of mechanical strain. Antibody microarrays of strained VSMC lysates revealed MEK1, MEK2, phospo-MEK1T385, T291, T298, phospho-Erk1/2T202+Y204/T185+T187, and PKC isoforms expression were universally increased, suggesting a proliferative/inflammatory signaling state. Conversely, VSMC strain decreased expression levels of Cdk1, Cdk2, Cdk4, and Cdk6 by 25-50% suggesting a partially inhibited proliferative signaling cascade. CONCLUSIONS: Subjecting VSMC to cyclic biomechanical strain in vitro promotes cell hypertrophy while attenuating cellular proliferation. We also report an upregulation of MEK and ERK activation suggestive of a proliferative phenotype. Hhowever, the proliferative response appears to be aborogated by enhanced antimitogenic cytokine VEGF, NO secretion and downregulation of Cdk expression. Although exogenous VEGF alone is not sufficient to promote the quiescent VSMC phenotype, we provide evidence suggesting that strain is a necessary component to induce VSMC response to the antimitogenic effects of VEGF. Taken together these data indicate that VEGF plays a critical role in mechanical strain-induced VSMC proliferation and vessel wall remodeling. Whether VEGF and/or NO inhibit signaling distal to Erk 1/2 is currently under investigation.