Dataset Information

Skeletal Muscle-Derived Human Mesenchymal Stem Cells: Influence of Different Culture Conditions on Proliferative and Myogenic Capabilities.

ABSTRACT: Skeletal muscle tissue is characterized by restrained self-regenerative capabilities, being ineffective in relation to trauma extension both in time span (e.g., chronic diseases) and in size (e.g., large trauma). For these reasons, tissue engineering and/or cellular therapies represent a valuable solution in the cases where the physiological healing process failed. Satellite cells, the putative skeletal muscle stem cells, have been the first solution explored to remedy the insufficient self-regeneration capacity. Nevertheless, some limitation related to donor age, muscle condition, expansion hitch, and myogenic potentiality maintenance have limited their use as therapeutic tool. To overcome this hindrance, different stem cells population with myogenic capabilities have been investigated to evaluate their real potentiality for therapeutic approaches, but, as of today, the perfect cell candidate has not been identified yet. In this work, we analyze the characteristics of skeletal muscle-derived human Mesenchymal Stem Cells (hMSCs), showing the maintenance/increment of myogenic activity upon differential culture conditions. In particular, we investigate the influence of a commercial enriched growth medium (Cyto-Grow), and of a medium enriched with either human-derived serum (H.S.) or human Platelet-rich Plasma (PrP), in order to set up a culture protocol useful for employing this cell population in clinical therapeutic strategies. The presented results reveal that both the enriched medium (Cyto-Grow) and the human-derived supplements (H.S. and PrP) have remarkable effects on hMSCs proliferation and myogenic differentiation compared to standard condition, uncovering the real possibility to exploit these human derivatives to ameliorate stem cells yield and efficacy.

SUBMITTER: Testa S

PROVIDER: S-EPMC7526461 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Skeletal Muscle-Derived Human Mesenchymal Stem Cells: Influence of Different Culture Conditions on Proliferative and Myogenic Capabilities.

Testa Stefano S Riera Carles Sánchez CS Fornetti Ersilia E Riccio Federica F Fuoco Claudia C Bernardini Sergio S Baldi Jacopo J Costantini Marco M Foddai Maria Laura ML Cannata Stefano S Gargioli Cesare C

Frontiers in physiology 20200916

Skeletal muscle tissue is characterized by restrained self-regenerative capabilities, being ineffective in relation to trauma extension both in time span (e.g., chronic diseases) and in size (e.g., large trauma). For these reasons, tissue engineering and/or cellular therapies represent a valuable solution in the cases where the physiological healing process failed. Satellite cells, the putative skeletal muscle stem cells, have been the first solution explored to remedy the insufficient self-rege ...[more]

PMID: 33041857

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Project description:BackgroundSkeletal muscle in livestock develops into meat, an important source of protein and other nutrients for human consumption. The muscle is largely composed of a fixed number of multinucleated myofibers determined during late gestation and remains constant postnatally. A population of postnatal muscle stem cells, called satellite cells, gives rise to myoblast cells that can fuse with the existing myofibers, thus increasing their size. This requires a delicate balance of transcription and growth factors and specific microRNA (miRNA) expressed by satellite cells and their supporting cells from the muscle stem cell niche. The role of transcription and growth factors in bovine myogenesis is well-characterized; however, very little is known about the miRNA activity during this process. We have hypothesized that the expression of miRNA can vary between primary cultures of skeletal muscle cells isolated from the semitendinosus muscles of different cattle breeds and subjected to myogenic differentiation.ResultsAfter a 6-day myogenic differentiation of cells isolated from the muscles of the examined cattle breeds, we found statistically significant differences in the number of myotubes between Hereford (HER)/Limousine (LIM) beef breeds and the Holstein-Friesian (HF) dairy breed (p ≤ 0.001). The microarray analysis revealed differences in the expression of 23 miRNA among the aforementioned primary cultures. On the basis of a functional analysis, we assigned 9 miRNA as molecules responsible for differentiation progression (miR-1, -128a, -133a, -133b, -139, -206, -222, -486, and -503). The target gene prediction and functional analysis revealed 59 miRNA-related genes belonging to the muscle organ development process.ConclusionThe number of myotubes and the miRNA expression in the primary cultures of skeletal muscle cells derived from the semitendinosus muscles of the HER/LIM beef cattle breeds and the HF dairy breed vary when cells are subjected to myogenic differentiation. The net effect of the identified miRNA and their target gene action should be considered the result of the breed-dependent activity of satellite cells and muscle stem cell niche cells and their mutual interactions, which putatively can be engaged in the formation of a larger number of myotubes in beef cattle-related cells (HER/LIM) during in vitro myogenesis.

Project description: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.

Dataset Information

Skeletal Muscle-Derived Human Mesenchymal Stem Cells: Influence of Different Culture Conditions on Proliferative and Myogenic Capabilities.

Publications

Skeletal Muscle-Derived Human Mesenchymal Stem Cells: Influence of Different Culture Conditions on Proliferative and Myogenic Capabilities.

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