3D skeletal muscle fascicle engineering is improved with TGF-β1 treatment of myogenic cells and their co-culture with myofibroblasts

Abstract

BackgroundSkeletal muscle wound healing is dependent on complex interactions between fibroblasts, myofibroblasts, myogenic cells, and cytokines, such as TGF-β1. This study sought to clarify the impact of TGF-β1 signaling on skeletal muscle cells and discern between the individual contributions of fibroblasts and myofibroblasts to myogenesis when in co-culture with myogenic cells. 3D tissue-engineered models were compared to equivalent 2D culture conditions to assess the efficacy of each culture model to predictively recapitulate the in vivo muscle environment.MethodsTGF-β1 treatment and mono-/co-cultures containing human dermal fibroblasts or myofibroblasts and C2C12 mouse myoblasts were assessed in 2D and 3D environments. Three culture systems were compared: cell monolayers grown on 2D dishes and 3D tissues prepared via a self-assembly method or collagen 1-based hydrogel biofabrication. qPCR identified gene expression changes during fibroblast to myofibroblast and myoblast differentiation between culture conditions. Changes to cell phenotype and tissue morphology were characterized via immunostaining for myosin heavy chain, procollagen, and α-smooth muscle actin. Tissue elastic moduli were measured with parallel plate compression and atomic force microscopy systems, and a slack test was employed to quantify differences in tissue architecture and integrity.ResultsTGF-β1 treatment improved myogenesis in 3D mono- and co-cultures containing muscle cells, but not in 2D. The 3D TGF-β1-treated co-culture containing myoblasts and myofibroblasts expressed the highest levels of myogenin and collagen 1, demonstrating a greater capacity to drive myogenesis than fibroblasts or TGF-β1-treatment in monocultures containing only myoblasts. These constructs possessed the greatest tissue stability, integrity, and muscle fiber organization, as demonstrated by their rapid and sustained shortening velocity during slack tests, and the highest Young’s modulus of 6.55 kPA, approximate half the stiffness of in situ muscle. Both self-assembled and hydrogel-based tissues yielded the most multinucleated, elongated, and aligned muscle fiber histology. In contrast, the equivalent 2D co-culture model treated with TGF-β1 completely lacked myotube formation through suppression of myogenin gene expression.DiscussionThese results show skeletal muscle regeneration can be promoted by treating myogenic cells with TGF-β1, and myofibroblasts are superior enhancers of myogenesis than fibroblasts. Critically, both TGF-β1 treatment and co-culturing skeletal muscle cells with myofibroblasts can serve as myogenesis accelerators across multiple tissue engineering platforms. Equivalent 2D culture systems cannot replicate these affects, however, highlighting a need to continually improve in vitro models for skeletal muscle development, discovery of therapeutics for muscle regeneration, and research and development of in vitro meat products.

Muscle tissue engineering in fibrous gelatin: implications for meat analogs

“…Our histology and mechanical testing suggest that although cultured meats based on BAOSMC or RbSkMC show some similarities to natural meats, further work with improved skeletal myoblast differentiation are required to more closely recapitulate the mature contractile architecture observed in natural muscle. Prior work by our laboratory and others demonstrated that recapitulating natural muscle phenotypes in culture required biomimetic culture conditions 36,57–63 that account for substrate stiffness 57 and biochemistry, 58 anisotropic muscle alignment, 36,62 and chemical factors secreted by supporting cell types. 63 For these reasons, recapitulating the nutritional profiles of meat will likely require building on bioengineering strategies 64 that account for multiscale engineering of genetic and epigenetic factors, as well as cell–material and cell–cell interactions that contribute to tissue development.…”

Tissue Engineering for Clean Meat Production

“…In addition to its central role in muscle development, TGF-β contributes to mature skeletal muscle mass, and is a key regulator of intramuscular fibrogenesis (Kollias and McDermott, 2008;Miao et al, 2016). In addition, it was shown that TGF-β1 suppressed myogenesis in 2D cultures, while enhancing myogenesis in 3D cultures, rendering it a valuable regulator of in-vitro muscle tissue development (Krieger et al, 2018). Myogenesis can be induced by substrate stiffness (Levy-Mishali et al, 2009;Syverud et al, 2014), IGFs and myogenic miRNA (Vitello et al, 2004;Yin et al, 2013), or by inhibition of anti-myogenic GFs produced by nearby cells (Christov et al, 2007;Yin et al, 2013).…”

“…Fibroblast and myofibroblasts (Baum and Duffy, 2011) are simple to isolate and grow, and have a short cell cycle. They were shown to promote vascularization and muscle development in tissue engineered constructs (Shandalov et al, 2014;Ciccone, 2015;Mackey et al, 2017;Krieger et al, 2018), and are considered to have an important role in clean meat production (Pandurangan and Kim, 2015). However, isolated fibroblasts are usually not very well defined, and are often comprised of several subpopulations with few specific markers (Chapman et al, 2016).…”

Cell-Based Fish: A Novel Approach to Seafood Production and an Opportunity for Cellular Agriculture

“…In the past 5 years, research focused on cell-based meats has accelerated from the tasting of the cell-cultured hamburger in 2013 (Zaraska, 2013) and early publications, including lifecycle assessments and basic research (Tuomisto and Teixeira de Mattos, 2011;Post, 2012Post, , 2014, to a growing start-up community, updated life cycle assessments, and publications focused on refining the technologies required to accelerate cellbased meat production (Tuomisto et al, 2014;Krieger et al, 2018;Rubio et al, 2019). To date, research decisions in cell-based meat production, such as selection of cell species and cell type have been largely driven by market size and environmental impact (Rodriguez-Fernandez, 2019), rather than suitability of cells species and types suitable for large scale bioreactor cultivation.…”