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

作　　者：Cong Li Rui Bi Lin Wang Yu-Hua Ma Yong-Gang Yao Ping Zheng 

机构地区：[1]State Key Laboratory of Genetic Resources and Evolution,Kunming Institute of Zoology,Chinese Academy of Sciences,Kunming,Yunnan 650201,China [2]Key Laboratory of Animal Models and Human Disease Mechanisms of Yunnan Province,Kunming Institute of Zoology,Chinese Academy of Sciences,Kunming,Yunnan 650201,China [3]Kunming College of Life Science,University of Chinese Academy of Sciences,Kunming,Yunnan 650204,China [4]National Resource Center for Non-Human Primates,National Research Facility for Phenotypic&Genetic Analysis of Model Animals(Primate Facility),Kunming Institute of Zoology,Chinese Academy of Sciences,Kunming,Yunnan 650107,China [5]KIZ/CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases,Kunming Institute of Zoology,Chinese Academy of Sciences,Kunming,Yunnan 650204,China

出　　处：《Zoological Research》2023年第6期1080-1094,共15页动物学研究（英文）

基　　金：supported by the Ministry of Science and Technology of China (2021YFF0702700,STI2030-Major Project2021ZD0200900);National Natural Science Foundation of China (U2102202,U1702284);Yunnan Province (202305AH340006)。

摘　　要：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.

关 键 词：Tree shrews Spermatogonial stem cells Nicotinamide riboside DNA damage Mitochondrial dysfunction 

分 类 号：Q95[生物学—动物学]