ANIMAL PROTEINS FROM STEM CELLS AS AN ALTERNATIVE TO REDUCE THE ECOLOGICAL AND CLIMATE IMPACT OF ANIMAL FARMING

Cultivated meat represents a transformative innovation in the agri-food sector, with the potential to decouple protein production from livestock farming and drastically reduce the environmental, ethical, and biosafety costs associated with conventional meat. However, the field remains constrained by major technical and economic challenges, most notably the reliance on expensive growth factor–based culture systems and the limited scalability of primary cells. In this thesis, I present a modular and fully engineered human induced pluripotent stem cell (hiPSC) platform, GERALT (Genetically Enhanced Renewal and Autonomous Lineage Transdifferentiation), optimized for the large-scale, serum-free production of skeletal muscle tissue, to pave the way to the construction of a molecular mechanism transferable to livestock animal cells. By integrating tools from synthetic biology, genome engineering, and stem cell technology, this work addresses key bottlenecks in both the expansion and differentiation phases of cellular agriculture. First, I developed a growth factor–independent culture system by constitutively expressing a stabilized version of NANOG from a genomic safe harbor locus. This strategy enables long-term maintenance of hiPSCs in chemically defined medium without TGFβ supplementation, reducing costs and enhancing scalability. Single-cell transcriptomic profiling confirmed the maintenance of pluripotency and repression of neuroectodermal drift under these minimal conditions after over 40 days of culture. In parallel, I optimized a cost-effective, home-brew medium compatible with feeder-free and weekend-free workflows, enabling robust culture and genome engineering in scalable formats. Next, I established an inducible post-transcriptional silencing platform based on artificial micro RNAs (miR-E) to suppress exogenous NANOG expression in a precise, reversible, and scalable manner. This circuit was coupled to the controlled expression of MYOD1 to enable forward programming into skeletal muscle. The resulting hiPSC line synchronously activates myogenesis and represses exogenous pluripotency upon doxycycline exposure, yielding >98% homogeneous skeletal myocytes in just 7 days. I further optimized the circuit architecture by embedding the miR-E element into a synthetic intron within the MYOD1 transcript, thereby minimizing potential interference between RNA processing and translation. Engineered cells were successfully adapted to suspension culture in defined media, maintaining pluripotency and forward programming capacity. Upon induction, they formed muscle fibers with robust expression of sarcomeric genes. The platform was also benchmarked for its metabolic robustness, demonstrating tolerance to variable glucose levels, a key parameter for industrial production. Finally, I streamlined the genetic engineering strategy into a single-locus, “three-in-one” construct combining inducible MYOD1, intronic miR-E, and a fluorescent reporter. We anticipate that this strategy will be translated to pig cells for cellular agriculture purposes. Altogether, this thesis establishes GERALT as a scalable and genetically encoded platform for growth factor–independent stem cell expansion and directed myogenesis, offering a concrete path toward industrially viable and species-adaptable cellular agriculture