Innovation in culture systems to study muscle complexity.

Endogenous skeletal muscle development, regeneration, and pathology are extremely complex processes, influenced by local and systemic factors. Unpinning how these mechanisms function is crucial for fundamental biology and to develop therapeutic interventions for genetic disorders, but also conditions like sarcopenia and volumetric muscle loss. Ex vivo skeletal muscle models range from two- and three-dimensional primary cultures of satellite stem cell-derived myoblasts grown alone or in co-culture, to single muscle myofibers, myobundles, and whole tissues. Together, these systems provide the opportunity to gain mechanistic insights of stem cell behavior, cell-cell interactions, and mature muscle function in simplified systems, without confounding variables. Here, we highlight recent advances (published in the last 5 years) using in vitro primary cells and ex vivo skeletal muscle models, and summarize the new insights, tools, datasets, and screening methods they have provided. Finally, we highlight the opportunity for exponential advance of skeletal muscle knowledge, with spatiotemporal resolution, that is offered by guiding the study of muscle biology and physiology with in silico modelling and implementing high-content cell biology systems and ex vivo physiology platforms.

Mini-MEndR: a miniaturized 96-well predictive assay to evaluate muscle stem cell-mediated repair.

Background: Functional evaluation of molecules that are predicted to promote stem cell mediated endogenous repair often requires in vivo transplant studies that are low throughput and hinder the rate of discovery. To offer greater throughput for functional validation studies, we miniaturized, simplified and expanded the functionality of a previously developed muscle endogenous repair (MEndR) in vitro assay that was shown to capture significant events of in vivo muscle endogenous repair. Methods: The mini-MEndR assay consists of miniaturized cellulose scaffolds designed to fit in 96-well plates, the pores of which are infiltrated with human myoblasts encapsulated in a fibrin-based hydrogel to form engineered skeletal muscle tissues. Pre-adsorbing thrombin to the cellulose scaffolds facilitates in situ tissue polymerization, a critical modification that enables new users to rapidly acquire assay expertise. Following the generation of the 3D myotube template, muscle stem cells (MuSCs), enriched from digested mouse skeletal muscle tissue using an improved magnetic-activated cell sorting protocol, are engrafted within the engineered template. Murine MuSCs are fluorescently labeled, enabling co-evaluation of human and mouse Pax7+ cell responses to drug treatments. A regenerative milieu is introduced by injuring the muscle tissue with a myotoxin to initiate endogenous repair "in a dish". Phenotypic data is collected at endpoints with a high-content imaging system and is analyzed using ImageJ-based image analysis pipelines. Results: The miniaturized format and modified manufacturing protocol cuts reagent costs in half and hands-on seeding time ~ threefold, while the image analysis pipelines save 40 h of labour. By evaluating multiple commercially available human primary myoblast lines in 2D and 3D culture, we establish quality assurance metrics for cell line selection that standardizes myotube template quality. In vivo outcomes (enhanced muscle production and Pax7+ cell expansion) to a known modulator of MuSC mediated repair (p38/ß MAPK inhibition) are recapitulated in the miniaturized culture assay, but only in the presence of stem cells and the regenerative milieu. Discussion: The miniaturized predictive assay offers a simple, scaled platform to co-investigate human and mouse skeletal muscle endogenous repair molecular modulators, and thus is a promising strategy to accelerate the muscle endogenous repair discovery pipeline. Supplementary Information: The online version contains supplementary material available at 10.1186/s44330-024-00005-4.

Three-dimensional niche stiffness synergizes with Wnt7a to modulate the extent of satellite cell symmetric self-renewal divisions.

Satellite cells (SCs), the resident adult stem cells of skeletal muscle, are required for tissue repair throughout life. While many signaling pathways are known to control SC self-renewal, less is known about the mechanisms underlying the spatiotemporal control of self-renewal during skeletal muscle repair. Here, we measured biomechanical changes that accompany skeletal muscle regeneration and determined the implications on SC fate. Using atomic force microscopy, we quantified a 2.9-fold stiffening of the SC niche at time-points associated with planar-oriented symmetric self-renewal divisions. Immunohistochemical analysis confirms increased extracellular matrix deposition within the basal lamina. To test whether three-dimensional (3D) niche stiffness can alter SC behavior or fate, we embedded isolated SC-associated muscle fibers within biochemically inert agarose gels tuned to mimic native tissue stiffness. Time-lapse microscopy revealed that a stiff 3D niche significantly increased the proportion of planar-oriented divisions, without effecting SC viability, fibronectin deposition, or fate change. We then found that 3D niche stiffness synergizes with WNT7a, a biomolecule shown to control SC symmetric self-renewal divisions via the noncanonical WNT/planar cell polarity pathway, to modify stem cell pool expansion. Our results provide new insights into the role of 3D niche biomechanics in regulating SC fate choice.

A 96-well culture platform enables longitudinal analyses of engineered human skeletal muscle microtissue strength.

Three-dimensional (3D) in vitro models of human skeletal muscle mimic aspects of native tissue structure and function, thereby providing a promising system for disease modeling, drug discovery or pre-clinical validation, and toxicity testing. Widespread adoption of this research approach is hindered by the lack of easy-to-use platforms that are simple to fabricate and that yield arrays of human skeletal muscle micro-tissues (hMMTs) in culture with reproducible physiological responses that can be assayed non-invasively. Here, we describe a design and methods to generate a reusable mold to fabricate a 96-well platform, referred to as MyoTACTIC, that enables bulk production of 3D hMMTs. All 96-wells and all well features are cast in a single step from the reusable mold. Non-invasive calcium transient and contractile force measurements are performed on hMMTs directly in MyoTACTIC, and unbiased force analysis occurs by a custom automated algorithm, allowing for longitudinal studies of function. Characterizations of MyoTACTIC and resulting hMMTs confirms the capability of the device to support formation of hMMTs that recapitulate biological responses. We show that hMMT contractile force mirrors expected responses to compounds shown by others to decrease (dexamethasone, cerivastatin) or increase (IGF-1) skeletal muscle strength. Since MyoTACTIC supports hMMT long-term culture, we evaluated direct influences of pancreatic cancer chemotherapeutics agents on contraction competent human skeletal muscle myotubes. A single application of a clinically relevant dose of Irinotecan decreased hMMT contractile force generation, while clear effects on myotube atrophy were observed histologically only at a higher dose. This suggests an off-target effect that may contribute to cancer associated muscle wasting, and highlights the value of the MyoTACTIC platform to non-invasively predict modulators of human skeletal muscle function.

A rapid in vitro methodology for simultaneous target discovery and antibody generation against functional cell subpopulations.

Cell surface antigen discovery is of great interest for biomedical research both for isolation of rare cell populations and therapeutic targeting. We developed a rapid, cost-effective, fully in vitro technology which facilities the simultaneous target discovery and human antibody generation on the surface of virtually any cell population of interest. We apply our technique to human colorectal cancer-initiating cells (CICs) and identify hundreds of unique human antibodies. We characterized the top three antibody candidates targeting these CICs and identify their protein targets as integrin α7 (ITGA7), HLA-A1 and integrin ß6 (ITGB6). We demonstrate that these antibodies can be used to isolate self-renewing colorectal CICs, and that the integrin α7 antibody can prospectively identify glioblastoma brain tumor initiating cells as well as human muscle stem cells. We also demonstrate that genetic ablation of integrin ß6 impedes colorectal CIC function. The methodology can be readily applied to other cell populations including stem cells, cancer, or immune cells to facilitate the rapid identification of novel targets and simultaneous generation of potent and specific antibodies with therapeutic potential.

Muscle stem cell intramuscular delivery within hyaluronan methylcellulose improves engraftment efficiency and dispersion.

Adult skeletal muscle tissue harbors the capacity for self-repair due to the presence of tissue resident muscle stem cells (MuSCs). Advances in the area of prospective MuSC isolation demonstrated the potential of cell transplantation therapy as a regenerative medicine strategy to restore strength and long-term regenerative capacity to aged, injured, or diseased skeletal muscle tissue. However, cell loss during ejection, limits to post-injection proliferation, and poor donor cell dispersion distal to the injection site are amongst hurdles to overcome to maximize MuSC transplant impact. Here, we assess a physical blend of hyaluronan and methylcellulose (HAMC) as a bioactive, shear thinning hydrogel cell delivery system to improve MuSC transplantation efficiency. Using in vivo transplantation studies, we found that the HAMC delivery system results in a >45% increase in the number of donor-derived fibers as compared to saline delivery. We demonstrate that increases in donor-derived fibers when using HAMC are attributed to increased MuSC proliferation via a CD44-independent mechanism, preventing injected cell active clearance, and supporting in vivo expansion by delaying differentiation. Furthermore, we observed a significant improvement in donor fiber dispersion when MuSCs were delivered in HAMC. Our study results suggest that HAMC is a promising muscle stem cell delivery vehicle.

Optimization of Satellite Cell Culture Through Biomaterials.

Hydrogels, a type of biomaterial, are an invaluable part of biomedical research as they are highly hydrated and properties such as elasticity, porosity, and ligand density can be tuned to desired values. Recently, culture substrate stiffness was found to be an important regulator of muscle stem cell self-renewal. Polyethylene glycol (PEG), a synthetic polymer, can be fabricated into hydrogels that match the softness of skeletal muscle tissue, thereby providing a culture surface that is optimal for maintaining muscle stem cell self-renewal potential ex vivo. In this Chapter, we describe a method to produce flat PEG hydrogels across a range of stiffnesses, including a formulation that matches the bulk stiffness of healthy skeletal muscle (12 kPa), while maintaining a constant ligand density. Since PEG is inert to protein adsorption, the steps required to surface functionalize the hydrogel with an adhesive interface (e.g., laminin) are also described.

Tethered Jagged-1 Synergizes with Culture Substrate Stiffness to Modulate Notch-Induced Myogenic Progenitor Differentiation.

INTRODUCTION: Notch signaling is amongst the key intrinsic mechanisms regulating satellite cell fate, promoting the transition of activated satellite cells to highly proliferative myogenic progenitor cells and preventing their premature differentiation. Although much is known about the biochemical milieu that drives myogenic progression, less is known about the spatial cues providing spatiotemporal control of skeletal muscle repair in the context of Notch signaling. METHODS: Using a murine injury model, we quantified in vivo biophysical changes that occur within the skeletal muscle during regeneration. Employing tunable poly(ethylene glycol)-based hydrogel substrates, we modeled the measured changes in bulk stiffness in the context of Notch ligand signaling, which are present in the regenerative milieu at the time of injury. RESULTS: Following injury, there is a transient increase in the bulk stiffness of the tibialis anterior muscle that may be explained in part by changes in extracellular matrix deposition. When presented to primary myoblasts, Jagged-1, Jagged-2, and Dll1 in a tethered format elicited greater degrees of Notch activity compared to their soluble form. Only tethered Jagged-1 effects were tuned by substrate stiffness, with the greatest Notch activation observed on stiff hydrogels matching the stiffness of regenerating muscle. When exposed to tethered Jagged-1 on stiff hydrogels, fewer primary myoblasts expressed myogenin, and pharmacological inhibitor studies suggest this effect is Notch and RhoA dependent. CONCLUSION: Our study proposes that tethered Jagged-1 presented in the context of transient tissue stiffening serves to tune Notch activity in myogenic progenitors during skeletal muscle repair and delay differentiation.

Biomechanical Origins of Muscle Stem Cell Signal Transduction.

Skeletal muscle, the most abundant and widespread tissue in the human body, contracts upon receiving electrochemical signals from the nervous system to support essential functions such as thermoregulation, limb movement, blinking, swallowing and breathing. Reconstruction of adult muscle tissue relies on a pool of mononucleate, resident muscle stem cells, known as "satellite cells", expressing the paired-box transcription factor Pax7 necessary for their specification during embryonic development and long-term maintenance during adult life. Satellite cells are located around the myofibres in a niche at the interface of the basal lamina and the host fibre plasma membrane (i.e., sarcolemma), at a very low frequency. Upon damage to the myofibres, quiescent satellite cells are activated and give rise to a population of transient amplifying myogenic progenitor cells, which eventually exit the cell cycle permanently and fuse to form new myofibres and regenerate the tissue. A subpopulation of satellite cells self-renew and repopulate the niche, poised to respond to future demands. Harnessing the potential of satellite cells relies on a complete understanding of the molecular mechanisms guiding their regulation in vivo. Over the past several decades, studies revealed many signal transduction pathways responsible for satellite cell fate decisions, but the niche cues driving the activation and silencing of these pathways are less clear. Here we explore the scintillating possibility that considering the dynamic changes in the biophysical properties of the skeletal muscle, namely stiffness, and the stretch and shear forces to which a myofibre can be subjected to may provide missing information necessary to gain a full understanding of satellite cell niche regulation.