A rapid and stable spontaneous reprogramming system of Spermatogonial stem cells to Pluripotent State

Rui Wei; Xiaoyu Zhang; Xiaoxiao Li; Jian Wen; Hongyang Liu; Jiqiang Fu; Li Li; Wenyi Zhang; Zhen Liu; Yang Yang; Kang Zou

doi:10.1186/s13578-023-01150-z

A rapid and stable spontaneous reprogramming system of Spermatogonial stem cells to Pluripotent State

Cell Biosci. 2023 Dec 1;13(1):222. doi: 10.1186/s13578-023-01150-z.

Authors

Rui Wei^#^{1

2}, Xiaoyu Zhang^#^{1

2}, Xiaoxiao Li^#^{1

2}, Jian Wen^{1

2}, Hongyang Liu^{1

2}, Jiqiang Fu³, Li Li³, Wenyi Zhang⁴, Zhen Liu³, Yang Yang⁵, Kang Zou^{6

7}

Affiliations

¹ Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China.
² Stem Cell Research and Translation Center, Nanjing Agricultural University, Nanjing, 210095, China.
³ Institute of Neuroscience, State Key Laboratory of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai, 200031, China.
⁴ State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, 211166, China.
⁵ State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, 211166, China. yangyang11@njmu.edu.cn.
⁶ Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China. kangzou@njau.edu.cn.
⁷ Stem Cell Research and Translation Center, Nanjing Agricultural University, Nanjing, 210095, China. kangzou@njau.edu.cn.

^# Contributed equally.

Abstract

Background: The scarcity of pluripotent stem cells poses a major challenge to the clinical application, given ethical and biosafety considerations. While germline stem cells commit to gamete differentiation throughout life, studies demonstrated the spontaneous acquisition of pluripotency by spermatogonial stem cells (SSCs) from neonatal testes at a low frequency (1 in 1.5 × 10⁷). Notably, this process occurs without exogenous oncogenes or chemical supplementation. However, while knockout of the p53 gene accelerates the transformation of SSCs, it also increases risk and hampers their clinical use.

Results: We report a transformation system that efficiently and stably convert SSCs into pluripotent stem cells around 10 passages with the morphology similar to that of epiblast stem cells, which convert to embryonic stem (ES) cell-like colonies after change with ES medium. Epidermal growth factor (EGF), leukemia inhibitory factor (LIF) and fresh mouse embryonic fibroblast feeder (MEF) are essential for transformation, and addition of 2i (CHIR99021 and PD0325901) further enhanced the pluripotency. Transcriptome analysis revealed that EGF activated the RAS signaling pathway and inhibited p38 to initiate transformation, and synergically cooperated with LIF to promote the transformation.

Conclusion: This system established an efficient and safe resource of pluripotent cells from autologous germline, and provide new avenues for regenerative medicine and animal cloning.

Keywords: Cell fate; Feeder layer; Germline; Pluripotency; Reprogramming.

Abstract

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