Age-related gene expression signatures from limb skeletal muscles and the diaphragm in mice and rats reveal common and species-specific changes.

BACKGROUND: As a result of aging, skeletal muscle undergoes atrophy and a decrease in function. This age-related skeletal muscle weakness is known as "sarcopenia". Sarcopenia is part of the frailty observed in humans. In order to discover treatments for sarcopenia, it is necessary to determine appropriate preclinical models and the genes and signaling pathways that change with age in these models. METHODS AND RESULTS: To understand the changes in gene expression that occur as a result of aging in skeletal muscles, we generated a multi-time-point gene expression signature throughout the lifespan of mice and rats, as these are the most commonly used species in preclinical research and intervention testing. Gastrocnemius, tibialis anterior, soleus, and diaphragm muscles from male and female C57Bl/6J mice and male Sprague Dawley rats were analyzed at ages 6, 12, 18, 21, 24, and 27 months, plus an additional 9-month group was used for rats. More age-related genes were identified in rat skeletal muscles compared with mice; this was consistent with the finding that rat muscles undergo more robust age-related decline in mass. In both species, pathways associated with innate immunity and inflammation linearly increased with age. Pathways linked with extracellular matrix remodeling were also universally downregulated. Interestingly, late downregulated pathways were exclusively found in the rat limb muscles and these were linked to metabolism and mitochondrial respiration; this was not seen in the mouse. CONCLUSIONS: This extensive, side-by-side transcriptomic profiling shows that the skeletal muscle in rats is impacted more by aging compared with mice, and the pattern of decline in the rat may be more representative of the human. The observed changes point to potential therapeutic interventions to avoid age-related decline in skeletal muscle function.

Population-scale analysis of common and rare genetic variation associated with hearing loss in adults.

To better understand the genetics of hearing loss, we performed a genome-wide association meta-analysis with 125,749 cases and 469,497 controls across five cohorts. We identified 53/c loci affecting hearing loss risk, including common coding variants in COL9A3 and TMPRSS3. Through exome sequencing of 108,415 cases and 329,581 controls, we observed rare coding associations with 11 Mendelian hearing loss genes, including additive effects in known hearing loss genes GJB2 (Gly12fs; odds ratio [OR] = 1.21, P = 4.2 × 10-11) and SLC26A5 (gene burden; OR = 1.96, P = 2.8 × 10-17). We also identified hearing loss associations with rare coding variants in FSCN2 (OR = 1.14, P = 1.9 × 10-15) and KLHDC7B (OR = 2.14, P = 5.2 × 10-30). Our results suggest a shared etiology between Mendelian and common hearing loss in adults. This work illustrates the potential of large-scale exome sequencing to elucidate the genetic architecture of common disorders where both common and rare variation contribute to risk.

A Microfluidic Eye Facsimile System to Examine the Migration of Stem-like Cells.

Millions of adults are affected by progressive vision loss worldwide. The rising incidence of retinal diseases can be attributed to damage or degeneration of neurons that convert light into electrical signals for vision. Contemporary cell replacement therapies have transplanted stem and progenitor-like cells (SCs) into adult retinal tissue to replace damaged neurons and restore the visual neural network. However, the inability of SCs to migrate to targeted areas remains a fundamental challenge. Current bioengineering projects aim to integrate microfluidic technologies with organotypic cultures to examine SC behaviors within biomimetic environments. The application of neural phantoms, or eye facsimiles, in such systems will greatly aid the study of SC migratory behaviors in 3D. This project developed a bioengineering system, called the µ-Eye, to stimulate and examine the migration of retinal SCs within eye facsimiles using external chemical and electrical stimuli. Results illustrate that the imposed fields stimulated large, directional SC migration into eye facsimiles, and that electro-chemotactic stimuli produced significantly larger increases in cell migration than the individual stimuli combined. These findings highlight the significance of microfluidic systems in the development of approaches that apply external fields for neural repair and promote migration-targeted strategies for retinal cell replacement therapy.

A novel electro-chemotactic approach to impact the directional migration of transplantable retinal progenitor cells.

Photoreceptor degeneration is a significant cause of visual impairment in the United States and globally. Cell replacement therapy shows great promise in restoring vision by transplanting stem-like cells into the sub-retinal space as substitutes for damaged photoreceptors. However, vision repair via transplantation has been limited, in large part, by low numbers of replacement cells able to migrate into damaged retinal tissue and integrate with native photoreceptors. Projects have used external chemical fields and applied electric fields to induce the chemotaxis and electrotaxis of replacement cells, respectively, with limited success. However, the application of combined electro-chemotactic fields in directing cells within biomaterials and host tissue has been surprisingly understudied. The current work examined the ability of combined electro-chemotactic fields to direct the migration of transplantable retinal progenitor cells (RPCs) in controlled microenvironments. Experiments used our established galvano-microfluidic system (Gal-MµS) to generate tunable chemotactic concentration fields with and without superimposed electric fields. Result illustrate that combination fields increased the distance migrated by RPCs by over three times that seen in either field, individually, and with greater directionality towards increasing gradients. Interestingly, immunofluorescence assays showed no significant differences in the distribution of the total and/or activated cognate receptor of interest, indicating that changes in ligand binding alone were not responsible for the measured increases in migration. Bioinformatics analysis was then performed to identity potential, synergistic mechanistic pathways involved in the electro-chemotaxis measured. Results indicate that increased RPC migration in electro-chemotactic fields may arise from down-regulation of cell adhesion proteins in tandem with up-regulation of cytoskeletal regulation proteins. These comprehensive results point towards a novel migration-targeted treatment that may dramatically improve transplantation outcomes as well as elucidate unreported synergy across biological mechanisms in response to electro-chemotactic fields.

Collective adhesion and displacement of retinal progenitor cells upon extracellular matrix substrates of transplantable biomaterials.

Strategies to replace retinal photoreceptors lost to damage or disease rely upon the migration of replacement cells transplanted into sub-retinal spaces. A significant obstacle to the advancement of cell transplantation for retinal repair is the limited migration of transplanted cells into host retina. In this work, we examine the adhesion and displacement responses of retinal progenitor cells on extracellular matrix substrates found in retina as well as widely used in the design and preparation of transplantable scaffolds. The data illustrate that retinal progenitor cells exhibit unique adhesive and displacement dynamics in response to poly-l-lysine, fibronectin, laminin, hyaluronic acid, and Matrigel. These findings suggest that transplantable biomaterials can be designed to improve cell integration by incorporating extracellular matrix substrates that affect the migratory behaviors of replacement cells.

A Gal-MµS Device to Evaluate Cell Migratory Response to Combined Galvano-Chemotactic Fields.

Electric fields have been studied extensively in biomedical engineering (BME) for numerous regenerative therapies. Recent studies have begun to examine the biological effects of electric fields in combination with other environmental cues, such as tissue-engineered extracellular matrices (ECM), chemical gradient profiles, and time-dependent temperature gradients. In the nervous system, cell migration driven by electrical fields, or galvanotaxis, has been most recently studied in transcranial direct stimulation (TCDS), spinal cord repair and tumor treating fields (TTF). The cell migratory response to galvano-combinatory fields, such as magnetic fields, chemical gradients, or heat shock, has only recently been explored. In the visual system, restoration of vision via cellular replacement therapies has been limited by low numbers of motile cells post-transplantation. Here, the combinatory application of electrical fields with other stimuli to direct cells within transplantable biomaterials and/or host tissues has been understudied. In this work, we developed the Gal-MµS device, a novel microfluidics device capable of examining cell migratory behavior in response to single and combinatory stimuli of electrical and chemical fields. The formation of steady-state, chemical concentration gradients and electrical fields within the Gal-MµS were modeled computationally and verified experimentally within devices fabricated via soft lithography. Further, we utilized real-time imaging within the device to capture cell trajectories in response to electric fields and chemical gradients, individually, as well as in combinatory fields of both. Our data demonstrated that neural cells migrated longer distances and with higher velocities in response to combined galvanic and chemical stimuli than to either field individually, implicating cooperative behavior. These results reveal a biological response to galvano-chemotactic fields that is only partially understood, as well as point towards novel migration-targeted treatments to improve cell-based regenerative therapies.

Predicted molecular signaling guiding photoreceptor cell migration following transplantation into damaged retina.

To replace photoreceptors lost to disease or trauma and restore vision, laboratories around the world are investigating photoreceptor replacement strategies using subretinal transplantation of photoreceptor precursor cells (PPCs) and retinal progenitor cells (RPCs). Significant obstacles to advancement of photoreceptor cell-replacement include low migration rates of transplanted cells into host retina and an absence of data describing chemotactic signaling guiding migration of transplanted cells in the damaged retinal microenvironment. To elucidate chemotactic signaling guiding transplanted cell migration, bioinformatics modeling of PPC transplantation into light-damaged retina was performed. The bioinformatics modeling analyzed whole-genome expression data and matched PPC chemotactic cell-surface receptors to cognate ligands expressed in the light-damaged retinal microenvironment. A library of significantly predicted chemotactic ligand-receptor pairs, as well as downstream signaling networks was generated. PPC and RPC migration in microfluidic ligand gradients were analyzed using a highly predicted ligand-receptor pair, SDF-1α - CXCR4, and both PPCs and RPCs exhibited significant chemotaxis. This work present a systems level model and begins to elucidate molecular mechanisms involved in PPC and RPC migration within the damaged retinal microenvironment.

A model microfluidics-based system for the human and mouse retina.

The application of microfluidics technologies to the study of retinal function and response holds great promise for development of new and improved treatments for patients with degenerative retinal diseases. Restoration of vision via retinal transplantation therapy has been severely limited by the low numbers of motile cells observed post transplantation. Using modern soft lithographic techniques, we have developed the µRetina, a novel and convenient biomimetic microfluidics device capable of examing the migratory behavior of retinal lineage cells within biomimetic geometries of the human and mouse retina. Coupled computer simulations and experimental validations were used to characterize and confirm the formation of chemical concentration gradients within the µRetina, while real-time images within the device captured radial and theta cell migration in response to concentration gradients of stromal derived factor (SDF-1), a known chemoattractant. Our data underscore how the µRetina can be used to examine the concentration-dependent migration of retinal progenitors in order to enhance current therapies, as well as develop novel migration-targeted treatments.

Dimensions of Biological Cellulose Nanocrystals Maximize Fracture Strength.

Cellulose nanocrystals (CNCs) exhibit outstanding mechanical properties exceeding that of Kevlar, serving as reinforcing domains in nature's toughest biological nanocomposites such as wood. To establish a molecular-level understanding of how CNCs develop high resistance to failure, here we present new analyses based on atomistic simulations on the fracture energy of Iß CNCs. We show that the fracture energy depends on the crystal width, due to edge defects that significantly reduce the fracture energy of small crystals but have a negligible effect beyond a critical width. Additionally, collective effects of sheet stacking and stabilization by van der Waals interactions saturate at a critical crystal thickness that we predict with an analytical relationship based on a physical model. Remarkably, ideal dimensions optimizing fracture energy are found to be 4.8-5.6 nm in thickness (approximately 6-7 layers) and 6.2-7.3 nm in width (approximately 6-7 cellulose chains), which correspond to the common dimensions of CNCs found in nature. Our studies shed light on evolutionary principles that provide guidance toward high mechanical performance in natural and synthetic nanobiocomposites.