Volume 44 Issue 6
Nov.  2023
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Cong Li, Rui Bi, Lin Wang, Yu-Hua Ma, Yong-Gang Yao, Ping Zheng. Characterization of long-term ex vivo expansion of tree shrew spermatogonial stem cells. Zoological Research, 2023, 44(6): 1080-1094. doi: 10.24272/j.issn.2095-8137.2023.317
Citation: Cong Li, Rui Bi, Lin Wang, Yu-Hua Ma, Yong-Gang Yao, Ping Zheng. Characterization of long-term ex vivo expansion of tree shrew spermatogonial stem cells. Zoological Research, 2023, 44(6): 1080-1094. doi: 10.24272/j.issn.2095-8137.2023.317

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

doi: 10.24272/j.issn.2095-8137.2023.317
RNA-seq data reported in this study were deposited in the Genome Sequence Archive database (http://gsa.big.ac.cn/) under Accession ID (CRA013123), in the Science Data Bank (https://www.scidb.cn/) under DOI: 10.57760/sciencedb.12594, and in the NCBI database under GSE228744.
Supplementary data to this article can be found online.
The authors declare that they have no competing interests.
C.L. performed most of the experiments and wrote the draft. R.B. and C.L. performed the transplantation assays. L.W. analyzed the RNA-seq data. Y.H.M. cared for the tree shrews. P.Z. and Y.G.Y. designed and supervised the study and revised the manuscript. All authors read and approved the final version of the manuscript.
Funds:  This work was supported by the Ministry of Science and Technology of China (2021YFF0702700, STI2030-Major Project 2021ZD0200900), National Natural Science Foundation of China (U2102202, U1702284), and Yunnan Province (202305AH340006)
More Information
  • 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.
  • RNA-seq data reported in this study were deposited in the Genome Sequence Archive database (http://gsa.big.ac.cn/) under Accession ID (CRA013123), in the Science Data Bank (https://www.scidb.cn/) under DOI: 10.57760/sciencedb.12594, and in the NCBI database under GSE228744.
    Supplementary data to this article can be found online.
    The authors declare that they have no competing interests.
    C.L. performed most of the experiments and wrote the draft. R.B. and C.L. performed the transplantation assays. L.W. analyzed the RNA-seq data. Y.H.M. cared for the tree shrews. P.Z. and Y.G.Y. designed and supervised the study and revised the manuscript. All authors read and approved the final version of the manuscript.
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