Srila Witsanu, Pangjantuk Amorn, Kunhorm Phongsakorn, Chaicharoenaudomrung Nipha, Noisa Parinya
Laboratory of Cell-Based Assays and Innovations, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima, 30000 Thailand.
Division of Biology, Faculty of Science and Technology, Rajamangala University of Technology, Thanyaburi, Pathum Thani, 12110 Thailand.
3 Biotech. 2025 Apr;15(4):92. doi: 10.1007/s13205-025-04225-5. Epub 2025 Mar 19.
Muscle satellite cells (MSCs) are essential for cultured meat production although their restricted lifespan and diminished stemness during prolonged culture pose significant limitations. This study established immortalized porcine MSCs using gene deletion with the CRISPR/Cas9 technology. Several -knockout (KO) clones were generated, exhibiting indel alterations and monoallelic and biallelic deletions. These clones exhibited markedly prolonged cellular lifespans compared to wild-type cells, overcoming the constraints of senescence. The growth rates and the proliferation marker (ki67) gene expression (passage 20) in the -KO clones were dramatically elevated compared to the WT cells. All -KO clones demonstrated a loss of stemness, proliferation, and muscle differentiation marker gene expression during long-term cell culture except for Desmin expression in the -KO 42 clone. Immortalized -KO clones maintained the ability to express muscle-specific protein markers compared to wild-type cells. Moreover, all clones had non-tumorigenic behavior, except -KO clones 41 and 42, which displayed tumorigenic potential. -KO clones demonstrated enhanced myogenic differentiation efficiency after multiple passages in comparison to wild-type cells. The results highlight the potential of -KO MSCs as a cellular resource for future cultured meat production.
The online version contains supplementary material available at 10.1007/s13205-025-04225-5.
肌肉卫星细胞（MSCs）对于培养肉的生产至关重要，尽管它们在长期培养过程中有限的寿命和减弱的干性构成了重大限制。本研究使用CRISPR/Cas9技术通过基因缺失建立了永生化猪MSCs。产生了几个基因敲除（KO）克隆，表现出插入缺失改变以及单等位基因和双等位基因缺失。与野生型细胞相比，这些克隆的细胞寿命显著延长，克服了衰老的限制。与野生型细胞相比，基因敲除克隆在第20代时的生长速率和增殖标志物（ki67）基因表达显著升高。除了基因敲除42克隆中的结蛋白表达外，所有基因敲除克隆在长期细胞培养过程中均表现出干性、增殖和肌肉分化标志物基因表达的丧失。与野生型细胞相比，永生化基因敲除克隆保持了表达肌肉特异性蛋白标志物的能力。此外，除了基因敲除克隆41和42具有致瘤潜力外，所有克隆均具有非致瘤行为。与野生型细胞相比，基因敲除克隆在多次传代后表现出增强的成肌分化效率。结果突出了基因敲除MSCs作为未来培养肉生产细胞资源的潜力。
在线版本包含可在10.1007/s13205-025-04225-5获取的补充材料。
