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

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12958-025-01389-w.