Characterization of long-term ex vivo expansion of tree shrew spermatogonial stem cells

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)-derivedspermatocytesor 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.

NAD＋ and its precursors in human longevity

Aging is a complex issue due to its nature in progressive physiological and functional decay. As better medicine, technology, and living conditions became accessible to many people, the longevity of human beings increased during the past centuries. Recent research established vital roles for NAD＋ and its precursors in protecting and maintaining the redox homeostasis in cells, which might be applicable therapeutically to prevent cell degeneration. Notably, the contribution of NAD＋ metabolites to lifespan extension in model systems indicates that the potential beneficial effects of NAD＋ precursors. In this mini review, by introducing the background of NAD＋-consuming enzymes in ＂caloric restriction＂, we focus on NAD＋ and its precursors in diet, with further emphasis on its association with health and diseases. We also provide insights in future utilization ofNAD＋ and its precursors as nutrition supplement for lifespan extension.