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Background <p>Testicular somatic cells play an important role in supporting spermatogenesis. Leydig cells (LCs) and peritubular myoid cells (PTMs) originate from a common progenitor population and show similar expression signatures in adulthood, making it difficult to distinguish and isolate the two in vitro.</p> Methods <p>In this study, new surface markers for identifying adult LCs (ALCs) and PTMs were discovered by reanalyzing testicular single-cell dataset. Differential expressions of ITGA9 and NGFR were confirmed through immunofluorescence staining of human testes. A novel Fluorescence activated Cell Sorting (FACS) protocol is established for the isolation of ALCs and PTMs based on the two markers. Long-term culture of both cells were performed and their characteristics were characterized and explored.</p> Results <p>ITGA9+ /NGFR + cells were positive for markers of PTMs (SMA, CNN1) and negative for markers of ALCs (HSD3B, STAR), and were able to form tubular and spheroid structures in vitro. In contrast, ITGA9-/NGFR + cells were positive for ALC markers and negative for PTM markers, and showed a capacity of testosterone production in vitro. Also, both cells were negative for Sertoli cell marker SOX9. When the two cells were cultured, they can expand for more than 15 passages.</p> Conclusions <p>Our study established a novel and efficient method for identifying and isolating human ALCs and PTMs, which provides a great potential for researches of the two cell types in human.</p>

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Identification and isolation of human testicular peritubular myoid cells and Leydig cells by a combination of ITGA9 and NGFR

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年5月31日

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  • Sha Han,
  • Liangyu Zhao,
  • Jiaqiang Luo,
  • Chenkun Shi,
  • Chencheng Yao,
  • Zhiyong Ji,
  • Junwei Xu,
  • Shuai Xu,
  • Ruhui Tian,
  • Erlei Zhi,
  • Yuhua Huang,
  • Xin Liu,
  • Yuchuan Zhou,
  • Zhi Zhou,
  • Zheng Li,
  • Peng Li

摘要

Background

Testicular somatic cells play an important role in supporting spermatogenesis. Leydig cells (LCs) and peritubular myoid cells (PTMs) originate from a common progenitor population and show similar expression signatures in adulthood, making it difficult to distinguish and isolate the two in vitro.

Methods

In this study, new surface markers for identifying adult LCs (ALCs) and PTMs were discovered by reanalyzing testicular single-cell dataset. Differential expressions of ITGA9 and NGFR were confirmed through immunofluorescence staining of human testes. A novel Fluorescence activated Cell Sorting (FACS) protocol is established for the isolation of ALCs and PTMs based on the two markers. Long-term culture of both cells were performed and their characteristics were characterized and explored.

Results

ITGA9+ /NGFR + cells were positive for markers of PTMs (SMA, CNN1) and negative for markers of ALCs (HSD3B, STAR), and were able to form tubular and spheroid structures in vitro. In contrast, ITGA9-/NGFR + cells were positive for ALC markers and negative for PTM markers, and showed a capacity of testosterone production in vitro. Also, both cells were negative for Sertoli cell marker SOX9. When the two cells were cultured, they can expand for more than 15 passages.

Conclusions

Our study established a novel and efficient method for identifying and isolating human ALCs and PTMs, which provides a great potential for researches of the two cell types in human.