Increased microenvironment stiffness in damaged myofibers promotes myogenic progenitor cell proliferation

Abstract

BackgroundThe stiffness of the myogenic stem cell microenvironment markedly influences the ability to regenerate tissue. We studied the effect of damaged myofibers on myogenic progenitor cell (MPC) proliferation and determined whether the structural integrity of the microenvironment contributes to phenotypic changes.MethodsIndividual myofibers were isolated and cultured for 6 days. During this period, the cytoskeleton of myofibers and transcription factors regulating MPC differentiation were characterized by immunostaining. Atomic Force Microscopy (AFM) was performed to measure stiffness of cultured myofibers. Healthy and damaged myofibers, and their associated MPCs, were studied in skeletal muscle from dystrophic and tenotomy mouse models. MPCs were cultured on stiffness-tunable substrates, and their phenotypes were assessed by immunostaining of myogenic transcription factors.ResultsWe showed that individual myofibers tend to shrink or collapse when cultured ex vivo starting from day 1 and that this is associated with a marked increase in the number of proliferative MPCs (Pax7+MyoD+). The myofibers collapsed due to a loss of viability as shown by Evans blue dye uptake and the disorganization of their cytoskeletons. Interestingly, collapsed myofibers in mdx skeletal muscles were similar to damaged myofibers in that they lose their viability, have a disorganized cytoskeleton (actin and α-actinin), and display local MPC (MyoD+) proliferation at their periphery. In a tenotomy model that causes loss of muscle tension, the cytoskeletal disorganization of myofibers also correlated with the activation/proliferation of MPCs. A deeper analysis of collapsed myofibers revealed that they produce trophic factors that influence MPC proliferation. In addition, collapsed myofibers expressed several genes related to the basal lamina. Immunostaining revealed the presence of fibronectin in the basal lamina and the cytoplasm of damaged myofibers. Lastly, using atomic force microscopy (AFM), we showed that collapsed myofibers exhibit greater stiffness than intact myofibers. Growing MPCs on a 2-kPa polyacrylamide-based substrate, exempt of additional microenvironmental cues, recapitulated proliferation and reduced spontaneous differentiation compared to growth on a 0.5-kPa substrate.ConclusionsOur results support the notion that collapsed or damaged myofibers increase the structural stiffness of the satellite cell microenvironment, which in addition to other cues such as trophic factors and changes in extracellular matrix composition, promotes the proliferation and maintenance of MPCs, required for myofiber repair.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0030-1) contains supplementary material, which is available to authorized users.

Laminin 521 maintains differentiation potential of mouse and human satellite cell-derived myoblasts during long-term culture expansion

“…Considering this, collagen VI in combination with laminin 521 may reduce the spontaneous differentiation during expansion, at which time collagen VI could be removed to induce differentiation. ECM stiffness is a critical regulator balancing myogenesis towards proliferation or differentiation [28–32]. Combining laminin 521 with MG or a hydrogel may provide increased performance in both proliferation and/or differentiation by providing a method to customize stiffness of the substrate to optimize both proliferation and differentiation conditions.…”

Mechanosensing of matrix by stem cells: From matrix heterogeneity, contractility, and the nucleus in pore-migration to cardiogenesis and muscle stem cells in vivo

“…Damage to myofibers in culture increases their stiffness and causes a marked increase in proliferative MuSCs [82]. Growing mouse MuSCs on hydrogels with matching stiffness recapitulated the proliferation of MuSCs, indicating mechanosensing as a key activator of MuSC in response to muscle fiber injury.…”

Erratum to: Increased microenvironment stiffness in damaged myofibers promotes myogenic progenitor cell proliferation

“…Following publication of the original article [1] it was brought to our attention that the fourth author’s surname had been misspelt. The author name was incorrectly written as Thomas Söllrald.…”