The global demand for meat is increasing due to population growth and changing dietary habits, placing immense pressure on the livestock industry and raising concerns about environmental sustainability, animal welfare, and public health. Cultured meat (CM), produced through the in vitro differentiation of stem cells, presents a potential solution. However, current CM production faces challenges, including the limited lifespan and differentiation capacity of commonly used muscle stem cell lines. This research aimed to address these challenges by utilizing porcine pre-gastrulation epiblast stem cells (pgEpiSCs), which possess the ability to self-renew indefinitely and differentiate into various cell types. The study also aimed to develop a serum-free myogenic differentiation system and a 3D tissue-shaping system using plant-based scaffolds to create meat-like tissue from pgEpiSCs. Successful development of these methods would significantly advance cultured meat production, offering a more sustainable and ethical alternative to traditional meat production.

Existing methods for cultured meat production often rely on muscle stem cells (MuSCs), which have limitations in long-term in vitro culture and differentiation. While immortalized cell lines have been explored, their regulatory mechanisms remain unclear. Pluripotent stem cells (PSCs), such as those derived from epiblasts, offer a promising alternative due to their self-renewal capacity and differentiation potential. However, establishing stable PSC lines from livestock species has been challenging. Previous research has explored various approaches for myogenic differentiation, including serum-dependent and transgenic methods. Serum-free systems are highly desirable for large-scale CM production, but developing such systems for livestock PSCs has proven difficult. Similarly, the development of suitable, animal-free 3D scaffolds is crucial for creating meat-like tissue with desirable textural properties. Existing plant-based scaffolds often require animal-derived components for optimal cell adhesion and growth.

The study began by validating the stability of previously established pgEpiSCs lines. Long-term in vitro culture, embryoid body (EB) differentiation assays, alkaline comet assays, and karyotype assays were used to assess their pluripotency, differentiation potential, and genomic stability. A serum-free myogenic differentiation system was established through a series of optimization steps. The researchers tested different combinations of growth factors and small molecules targeting signaling pathways involved in mesoderm and myogenic differentiation. The efficacy of the differentiation system was evaluated by immunostaining for key marker proteins, flow cytometry, RNA sequencing (RNA-seq), and untargeted metabolomics analysis. Plant-based 3D edible scaffolds were developed using konjac glucomannan (KGM) and sodium alginate (SA), cross-linked with calcium ions. The optimal scaffold composition was determined based on its structural properties, cell adhesion, survival, water absorption, degradation rate, and mechanical stability. pgEpiSCs were differentiated into myoblasts and subsequently cultured on the chosen scaffold to generate 3D meat-like tissue. The resulting tissue was assessed for cell survival, proliferation, morphology, gene expression, amino acid composition, and texture. Microbial contamination was also checked. Standard techniques such as immunofluorescence, western blotting, qPCR, RNA-Seq, and metabolomic analyses were employed to assess cellular characteristics and differentiation success.

The study demonstrated the long-term stability and pluripotency of pgEpiSCs, which maintained their genomic integrity and differentiation potential even after 200 passages. A transgene-free and serum-free myogenic differentiation protocol was successfully established, inducing pgEpiSCs into mature skeletal muscle fibers (SMFs). RNA-seq analysis confirmed the expected lineage progression at each stage of differentiation, revealing a distinct transcriptional profile for pgEpiSCs-derived SMFs compared to pgEpiSCs and porcine embryonic fibroblasts. Untargeted metabolomics analysis revealed significant metabolic changes during myogenic differentiation, including alterations in pathways related to vitamin B6 metabolism, the pentose phosphate pathway, and energy metabolism. Plant-based 3D edible scaffolds composed of KGM and SA were successfully developed, providing a suitable environment for pgEpiSCs-derived myoblast attachment, proliferation, and differentiation into a 3D meat-like tissue structure. The generated cultured meat exhibited desirable textural properties and was rich in essential amino acids, while being free from foodborne pathogenic bacteria. The pgEpiSCs-derived cultured meat had comparable transcriptomic and metabolomic characteristics to those from porcine MuSCs.

This study successfully addressed several key challenges in cultured meat production. The use of stable pgEpiSCs as the starting cell line eliminates the limitations associated with MuSCs and provides a readily scalable cell source. The development of a serum-free differentiation system reduces costs and improves the safety of the process. The use of a plant-based scaffold removes reliance on animal-derived components, addressing ethical concerns and enhancing the sustainability of cultured meat production. The generation of meat-like tissue with desirable texture and nutritional profile represents a significant step toward commercially viable cultured meat production. The findings align well with the growing body of research demonstrating the potential of PSCs for cellular agriculture. Further research should focus on optimizing the 3D scaffold design to enhance textural properties, simplifying the differentiation protocol for large-scale manufacturing, and reducing the cost of culture media components.

This research demonstrates a significant advancement in cultured meat production by using stable pgEpiSCs and developing a serum-free differentiation system and a plant-based 3D scaffold. The resulting cultured meat shows promise as a sustainable and ethical alternative to traditional meat. Future research should focus on scaling up the production process, optimizing the 3D scaffold's mechanical and textural properties, and further reducing the cost of production to enhance its commercial viability.

The study's limitations include the relatively small scale of 3D tissue production. Scaling up to commercially relevant production volumes will require further optimization of the process. The cost of some of the culture media components, particularly the growth factors, remains a factor that needs to be addressed for wider adoption. Long-term studies on the safety and nutritional value of this cultured meat are also warranted before its widespread consumption.