Efficient in vitro generation of skeletal muscle tissue from cell lines is pivotal for the advancement of cultivated meat production. Here, we introduce a novel system employing a synthetic temperature-sensitive promoter (HSP70) carrying multiple heat shock elements (HSE), whose activity can be modulated by changes in temperature, enabling precise control of the expression of an exogenous master gene of myogenesis (MyoD) and a chromatin modifier (SMARCD3). To establish the optimal conditions, we screened different versions of temperature-inducible promoters (with 5, 15, or 45 HSE repetitions) in mammalian cell lines. Additionally, we explored the concurrent overexpression of exogenous heat shock factor 1 (HSF1), a multifaceted factor that enhances Hsp70 transcription, regulates the localization of binding partners, and modulates translation under stress. Our results demonstrate efficient induction of exogenous pro-myogenic factors with a 2-hour heat shock at 43°C. In the presence of exogenous HSF1, we observed a rapid initiation of the myogenic differentiation process and a significant acceleration in the expression of late myogenic markers (myogenin and sarcomeric myosin heavy chain), as well as the formation of skeletal muscle fibers in cells with intrinsic myogenic potential, such as L6 myoblasts. This approach allows for precise regulation of gene expression and cellular responses, leading to rapid and efficient production of mature muscle fibers without the need for expensive media formulations or genetic-based drug-inducible systems. This temperature-dependent cellular engineering method offers significant advantages, including accurate spatial and temporal control over cell myogenic maturation and scalability for large-scale production of differentiated skeletal muscle tissue for cultivated meat production.
| Identifer | oai:union.ndltd.org:unitn.it/oai:iris.unitn.it:11572/427490 |
| Date | 28 October 2024 |
| Creators | Fioravanti, Giulia |
| Contributors | Fioravanti, Giulia, Biressi, Stefano Augusto Maria, Conti, Luciano |
| Publisher | Università degli studi di Trento, place:TRENTO |
| Source Sets | Università di Trento |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis |
| Rights | info:eu-repo/semantics/embargoedAccess |
| Relation | firstpage:1, lastpage:128, numberofpages:128 |
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