Biology subjects

Jacques, E.

Publications and source records attributed to Jacques, E..

3 recordsLinked to original sources

bioRxiv2023

Electron microscopic analysis of the influence of iPSC-derived motor neurons on bioengineered human skeletal muscle tissues

3D bioengineered skeletal muscle macrotissues are increasingly important for studies of cell biology and development of therapeutics. Tissues derived from immortalized cells obtained from patient samples or from stem cells can be co-cultured with motor-neurons to create models of human neuromuscular junctions in culture. In this study, we present foundational work on 3D cultured muscle ultrastructure, with and without motor neurons, which is enabled by the development of a new co-culture platform. Our results show that tissues from Duchenne muscular dystrophy patients are poorly organized compared to tissues grown from healthy donor and that the presence of motor neurons invariably improves sarcomere organization. Electron micrographs show that in the presence of motor neurons, filament directionality, banding patterns, z-disc continuity and appearance of presumptive SSR and T-tubule profiles all improve in healthy, DMD and iPSC derived muscle tissue. Further work to identify the underlying defects of DMD tissue disorganization and the trophic mechanisms by which motor neurons support muscle are likely to yield potential new therapeutic approaches for treating patients suffering from Duchenne muscular dystrophy.

cell biology↗

bioRxiv2023

mini-MEndR: A miniaturized 96-well predictive assay to evaluate muscle stem cell mediated repair

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. Here, we offer a strategy to rapidly test and prioritize molecules 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 the first week of the in vivo muscle endogenous repair process. The new "mini-MEndR assay" consists of miniaturized cellulose scaffolds designed to fit in 96-well plates. The scaffold pores are infiltrated with myoblasts encapsulated in a fibrin-based hydrogel to form thin, engineered skeletal muscle tissues. By evaluating multiple commercially available human primary myoblast lines in 2D and 3D culture, we establish quality assurance metrics for cell line selection that standardize myotube template quality. Pre-adsorbing thrombin to the cellulose scaffolds facilitates in situ tissue polymerization, a critical modification that enables users proficient in myoblast culture to rapidly acquire myotube template fabrication 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 onto the engineered human muscle template. A regenerative milieu is then introduced by injuring the muscle tissue with a myotoxin. Addition of a known modulator of MuSC mediated repair recapitulates the in vivo outcomes (enhanced muscle production and Pax7+ cell expansion), but only in the presence of both the stem cells and the regenerative milieu. By fluorescently labeling the mouse MuSCs, we demonstrate the feasibility of co-evaluating human and mouse Pax7+ cell responses to drug treatment, thereby expanding the utility of the assay. Importantly, phenotypic data is collected with a high-content imaging system and is analyzed using CellProfiler-based image analysis pipelines. The miniaturized predictive assay offers a simple, scaled platform with which 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.

bioengineering↗

bioRxiv2022

Rescue of aged muscle stem cell intrinsic quiescence defects by AKT inhibition revealed with a 3D biomimetic culture assay

Adult skeletal muscle harbors a population of muscle stem cells (MuSCs) that are required to repair or reform multinucleated myofibers after tissue injury. In youth, MuSCs return to a reversible state of cell cycle arrest termed quiescence after injury resolution. By contrast, a proportion of MuSCs in aged muscle remain in a semi-activated state, causing a premature response to subsequent injury cues that results in incomplete tissue repair and eventual stem cell depletion. Regulation of the balance between MuSC quiescence and activation in youth and in age may hold the key to restoring tissue homeostasis with age, but is incompletely understood. To fill this gap, we developed a simple and tractable in vitro method, with a 96-well footprint, to rapidly inactivate MuSCs freshly isolated from young skeletal muscle tissue, and return them to a quiescent-like state for at least one-week. This was achieved by introducing MuSCs into a three-dimensional (3D) bioartificial niche comprised of a thin sheet of multinucleated myotubes, which we iterate, and analyze temporally, to show that these in vivo niche features provide the minimal cues necessary to inactivate MuSCs and induce quiescence. By seeding the 3D myotube sheets with different starting numbers of MuSCs, the assay revealed cellular heterogeneity and population-level adaptation activities that converged on a common steady-state niche repopulation density; behaviors previously observed only in vivo. Quiescence-associated hallmarks included a Pax7+CalcR+MyoD- c-FOS- molecular signature, in vivo quiescent-like morphologies including oval-shaped nuclei and long cytoplasmic projections with N-cadherin+ tips, as well as the acquisition of polarized niche markers. Leveraging high-content imaging and bespoke CellProfilerTM-based image analysis pipelines, we demonstrate a relationship between morphology and cell fate signatures opening up the possibility of real-time morphology-based screening. Notably, when MuSCs from aged muscle were introduced into the assay, they displayed aberrant proliferative activities, delayed inactivation kinetics, persistence of activation-associated morphologies, and population depletion; quiescence-associated defects that we show are rescued by wortmannin treatment. Thus, the miniaturized assay offers an unprecedented opportunity to systematically investigate long-standing queries in areas such as regulation of adult stem cell pool size and functional heterogeneity within the MuSC population, and to uncover regulators of quiescence in youth and in age.

bioengineering↗