Enhanced Stem Cell Sorting Method Boosts Cultured Meat Potential

By optimizing cell surface marker combinations, the study significantly improves stem cell purity and myogenic potential during passaging, offering a robust source of seed cells essential for advancing cultured meat technologies.

Cultured meat technology, which cultivates animal-derived stem cells into edible meat products, presents an eco-friendly alternative to traditional livestock farming. However, significant challenges remain in obtaining and maintaining high-quality seed cells.MuSCs are pivotal for generating meat fibers, but isolating pure populations from complex muscle tissue has been difficult. Furthermore, standard cell sorting methods often result in declining stemness and differentiation ability during cell expansion. Addressing these limitations is crucial for producing viable, scalable cultured meat products. Based on these challenges, there is an urgent need to develop improved strategies for isolating and maintaining functional muscle stem cells for cultured meat production.

A study (DOI: 10.48130/fmr-0025-0001) published in Food Materials Research on 28 February 2025 by Renpeng Guo, Shijie Ding and Guanghong Zhou's team, Nanjing Agricultural University, offers a reliable and scalable method to produce high-quality seed cells crucial for cultured meat manufacturing.

To optimize the isolation of porcine MuSCs, researchers initially applied Ding's method, using enzymatic digestion to obtain mononuclear cells from piglet muscle tissue, followed by staining with fluorescent antibodies (CD31, CD45, CD56, and CD29) and fluorescence-activated cell sorting (FACS) to isolate CD31⁻/CD45⁻/CD29⁺/CD56⁺ MuSCs. Although this method initially achieved an 85% PAX7-positive cell population, successive passaging led to a dramatic decrease in stemness, with PAX7 expression dropping to 16.7% and myogenic fusion capacity declining to about 33% by passage five (P5). To address these limitations, a novel strategy incorporating CD31, CD45, JAM1, ITGA5, and ITGA7 markers was developed. By gating CD31⁻/CD45⁻/JAM1⁻ cells and further subdividing based on ITGA5 and ITGA7 expression, three populations were identified: ITGA5⁺/ITGA7⁻ (FAPs), ITGA5⁺/ITGA7⁺ (SMCs), and ITGA5⁻/ITGA7⁺ (MuSCs). Immunofluorescence, qPCR, and Western blot analyses revealed that the refined method achieved higher purity, with over 90% PAX7-positive MuSCs, enhanced PDGFRA and CNN1 marker specificity for FAPs and SMCs, respectively, and significantly more uniform cell morphology. Transcriptome sequencing further validated these identities, confirming distinct gene expression profiles and significant enrichment in skeletal muscle development pathways for the MuSC-enriched group. Functionally, the 5⁻7⁺ MuSCs exhibited superior differentiation potential, achieving a 90% myotube fusion rate compared to 61% using the conventional method, and maintained higher PAX7 expression and lower contaminating cell markers across multiple passages. Collectively, this novel sorting strategy significantly improves the isolation, expansion, and myogenic performance of porcine MuSCs, providing a more robust cell source for cultured meat production.

In summary, this study lays a critical foundation for the large-scale production of cultured pork, paving the way for sustainable, ethical, and high-quality meat alternatives.