Abstract
Chinese Tree shrews ( Tupaia belangeri chinensis) share a close relationship to primates and have been widely used in biomedical research. We previously established a spermatogonial stem cell (SSC)-based gene editing platform to generate transgenic tree shrews. However, the influences of long-term expansion on tree shrew SSC spermatogenesis potential remain unclear. Here, we examined the in vivo spermatogenesis potential of tree shrew SSCs cultured across different passages. We found that SSCs lost spermatogenesis ability after long-term expansion (>50 passages), as indicated by the failure to colonize the seminiferous epithelium and generate donor spermatogonia (SPG)-derived spermatocytes or spermatids marking spermatogenesis. RNA sequencing (RNA-seq) analysis of undifferentiated SPGs across different passages revealed significant gene expression changes after sub-culturing primary SPG lines for more than 40 passages on feeder layers. Specifically, DNA damage response and repair genes (e.g., MRE11, SMC3, BLM, and GEN1) were down-regulated, whereas genes associated with mitochondrial function (e.g., NDUFA9, NDUFA8, NDUFA13, and NDUFB8) were up-regulated after expansion. The DNA damage accumulation and mitochondrial dysfunction were experimentally validated in high-passage cells. Supplementation with nicotinamide adenine dinucleotide (NAD +) precursor nicotinamide riboside (NR) exhibited beneficial effects by reducing DNA damage accumulation and mitochondrial dysfunction in SPG elicited by long-term culture. Our research presents a comprehensive analysis of the genetic and physiological attributes critical for the sustained expansion of undifferentiated SSCs in tree shrews and proposes an effective strategy for extended in vitro maintenance. 树鼩（ Tupaia belangeri chinensis）与灵长类动物亲缘关系近，目前已被广泛应用于生物医学研究中。我们前期成功建立了树鼩精原干细胞（Spermatogonial stem cell, SSC）的体外培养体系，并基于此建立了国际首例转基因树鼩。然而，长期体外培养对树鼩精原干细胞生精潜能的影响尚不清楚，需要阐明。在本研究中，我们检测了体外培养的不同代数的树鼩精原干细胞的体内生精潜能。结果发现，长期体外扩增（大于50代）的细胞失去了移植后精子发生的能力，表现为不能定植在曲细精管，并产生供体细胞来源的精母细胞或精子。通过对不同代数细胞的转录组表达谱分析发现，随着体外培养时间的延长，基因表达发生显著变化。DNA损伤应答和修复基因（如 MRE11, SMC3, BLM, GEN1）在高代数培养后下调，而与线粒体功能相关的基因（如 NDUFA9, NDUFA8, NDUFA13, NDUFB8）在高代数培养后上调。通过对不同代数的细胞开展功能实验，发现高代数细胞呈现DNA损伤积累和线粒体功能改变。我们进一步发现NAD +前体烟酰胺核苷（Nicotinamide riboside, NR）的补充，对树鼩精原干细胞的长期培养有益。补充NR可降低长期培养引起的精原细胞DNA损伤积累和线粒体功能障碍。综上所述，该研究明确了长时间体外培养对树鼩精原干细胞功能的影响，并提出了培养体系改善的策略。.