MEndR: An In Vitro Functional Assay to Predict In Vivo Muscle Stem Cell‐Mediated Repair

Davoudi, Sadegh; Jacques, Eric; Cadavid, Jose L.; McFee, Matthew; Chin, Chih-Ying; Meysami, Amir; Ebrahimi, Majid; Bakooshli, Mohsen Afshar; Tung, Kayee; Ahn, Henry; Ginsberg, Howard J.; McGuigan, Alison P.; Gilbert, Penney M.

doi:10.1002/adfm.202106548

Cited by 18 publications

(46 citation statements)

References 58 publications

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“…Our study demonstrates the utility of myoscaffolds as a novel tool to study cell-ECM interactions and to test the efficacy of cell-based therapies, building on other elegant systems 83,84 . Several important biological components of the ECM, including receptor binding sites and mechanical properties, cannot be evaluated or replicated in standard culture dishes or in engineered matrices that lack native architecture and composition.…”

Section: Discussionmentioning

confidence: 88%

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

Stearns-Reider

Hicks

Hammond

et al. 2022

Preprint

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We developed an on-slide decellularization approach to generate acellular extracellular matrix (ECM) scaffolds 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.

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Section: Discussionmentioning

confidence: 88%

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

Stearns-Reider

Hicks

Hammond

et al. 2022

Preprint

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“…We previously reported a method to study skeletal muscle endogenous repair "in a dish" in a 24-well format by introducing MuSCs into thin sheets of engineered muscle tissue that we then injured using myotoxins. 33 In our uninjured control tissues, we observed a non-negligible proportion of the engrafted cells remained mononucleated at the assay endpoint, in spite of the differentiation-inducing culture media used. From this, we hypothesized that the muscle tissues were providing a pro-quiescence niche.…”

Section: Introductionmentioning

confidence: 64%

“…We previously reported a method to prepare thin sheets of human myotubes situated within a 24-well format, together with a strategy to evaluate mouse MuSC endogenous repair 'in a dish'. 33 Herein we adapted and extended the method to create thin sheets of murine myotubes that fit within a 96-well plate footprint. Briefly, we incorporated primary mouse myoblasts within a mixture of media, fibrinogen, and Geltrex TM (i.e.…”

Section: Engineered Myotube Templates Derived From Primary Mouse Myob...mentioning

confidence: 99%

“…Primary mouse MuSCs were isolated from mouse hindlimb muscle using a modified method previously reported by our group. 33 Briefly, ≈1 gram of muscle tissues was dissected from the hindlimb muscles of a humanely euthanized mouse and placed into a GentleMACS dissociation tube (Miltenyi Biotec, #130-096-334). 7 mL of DMEM (Gibco, #11995-073) with 630 U/mL Type 1A collagenase from clostridium histolyticum (Sigma, #C9891) was added to the tube, and the sample was physically dissociated using a GentleMACS dissociator (Miltenyi Biotec, #130-096-334) using the "skeletal muscle" setting.…”

Section: Magnetic-activated Cell Sorting (Macs) Of Primary Mouse Musc...mentioning

confidence: 99%

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

Jacques

Kuang

Kann

et al. 2022

Preprint

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Adult skeletal muscle harbors a population of muscle stem cells (MuSCs) that are required to repair or reform multinucleated myofibers after a tissue injury. In youth, a portion of MuSCs return to a reversible state of cell cycle arrest, termed quiescence, after injury resolution. By contrast, a proportion of aged MuSCs exist in a semi-activated state under homeostatic conditions, and prematurely respond to subsequent injury cues, thereby failing to return the tissue to its pre-injury state. The heterogeneity of MuSC function is linked to quiescence depth, but regulation of the balance between MuSC quiescence and activation in youth and in age is incompletely understood. This is due in part to the paucity of scalable methods that support MuSC quiescence in culture, and in turn necessitates reliance on low-throughput in vivo studies. To fill this gap, we developed a simple, 96-well format method to inactivate MuSCs isolated from skeletal muscle tissue, and return them to a quiescent-like state for at least one-week by culturing them within a three-dimensional engineered sheet of myotubes. Seeding the myotube sheets with different numbers of MuSCs elicited population-level adaptation activities that converged on a common steady-state niche repopulation density. By evaluating MuSC engraftment over time in culture, we observed reversible cell cycle exit that required both myotubes and a 3D culture environment. Additional quiescence-associated hallmarks were identified including a Pax7+CalcR+MyoD-c-FOS- molecular signature, quiescent-like morphology including oval-shaped nuclei and long cytoplasmic projections with N-cadherin+ tips, as well as the acquisition of polarized niche markers. We further demonstrate a relationship between morphology and cell fate signature using high-content imaging and CellProfilerTM-based image analysis pipelines. MuSC functional heterogeneity during engraftment was observed across all metrics tested, suggesting in vivo-like subpopulation activities are reflected in the assay. Notably, aged MuSCs introduced into young 3D myotube cultures displayed aberrant proliferative activities, delayed inactivation kinetics, and activation-associated morphologies that we show are rescued by wortmannin treatment. Thus, this miniaturized, biomimetic culture assay offers an unprecedented opportunity to uncover regulators of quiescence in youth and in age.

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“…Briefly, hADSCs were suspended in a fibrin/Geltrex™ hydrogel and pipetted onto the surface of a dry paper scaffold (Supplementary Information Figure 1). The fibrin/Geltrex™ hydrogel formulation was selected based on our previous use for other non-diseased human cell types 21,30 . Upon contact with the paper surface, the hydrogel-cell mixture infiltrated in between the paper fibers of the highly porous scaffold via capillary wicking.…”

Section: Optimization Of Human Adsc Infiltration and Differentiation ...mentioning

confidence: 99%

A three-dimensional human adipocyte model of fatty acid-induced obesity

Pieters¹,

Rjaibi²,

Singh³

et al. 2022

Biofabrication

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Obesity prevalence has reached pandemic proportions, leaving individuals at high risk for the development of diseases such as cancer and type 2 diabetes. In obesity, to accommodate excess lipid storage, adipocytes become hypertrophic, which is associated with an increased pro-inflammatory cytokine secretion and dysfunction of metabolic processes such as insulin signaling and lipolysis. Targeting adipocyte dysfunction is an important strategy to prevent the development of obesity-associated disease. However, it is unclear how accurately animal models reflect human biology, and the long-term culture of human hypertrophic adipocytes in an in vitro 2D monolayer is challenging due to the buoyant nature of adipocytes. Here we describe the development of a human 3D in vitro disease model that recapitulates hallmarks of obese adipocyte dysfunction. First, primary human adipose-derived mesenchymal stromal cells are embedded in hydrogel, and infiltrated into a thin cellulose scaffold. The thin microtissue profile allows for efficient assembly and image-based analysis. After adipocyte differentiation, the scaffold is stimulated with oleic or palmitic acid to mimic caloric overload. Using functional assays, we demonstrated that this treatment induced important obese adipocyte characteristics such as a larger lipid droplet size, increased basal lipolysis, insulin resistance and a change in macrophage gene expression through adipocyte-conditioned media. This 3D disease model mimics physiologically relevant hallmarks of obese adipocytes, to enable investigations into the mechanisms by which dysfunctional adipocytes contribute to disease.

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MEndR: An In Vitro Functional Assay to Predict In Vivo Muscle Stem Cell‐Mediated Repair

Cited by 18 publications

References 58 publications

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

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

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

A three-dimensional human adipocyte model of fatty acid-induced obesity

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