Integrative Machine Learning and Spatial Transcriptomic Analysis Reveals Vimentin as a Hub Gene Governing Spermatogonial Stem Cell Differentiation

Integrative Machine Learning and Spatial Transcriptomic Analysis Reveals Vimentin as a Hub Gene Governing Spermatogonial Stem Cell Differentiation

Abstract

Background: Spermatogenesis is a tightly regulated developmental process that sustains male fertility through the balance of spermatogonial stem cell (SSC) self-renewal and germ cell differentiation. Although recent single-cell studies have revealed cellular heterogeneity within the human testis, the coordinated transcriptional, epigenetic, and spatial regulation of key stemness and germ cell regulators during spermatogenesis remains incompletely understood.

Methods: Here, we performed an integrative multi-omic analysis combining single-cell RNA sequencing, DNA methylation profiling, pathway activity inference, pseudotime trajectory reconstruction, spatial transcriptomic integration, and immunofluorescence validation. We focused on a core regulatory network involving VIM, OCT4 (POU5F1), SOX2, KLF4, and DAZL to characterize their dynamic regulation across germ cell states.

Results: Single-cell transcriptomic analysis identified distinct germ cell populations spanning spermatogonial stem cells to spermatids and revealed progressive downregulation of pluripotency-associated transcription factors during differentiation. DNA methylation analysis demonstrated promoter hypermethylation of OCT4, SOX2, and KLF4 in spermatids, consistent with transcriptional silencing, while DAZL showed sustained expression and distinct epigenetic regulation in differentiating germ cells. Pseudotime and Gene Set Variation Analysis uncovered coordinated temporal shifts in stemness, cell cycle, apoptosis, and differentiation-related pathways. Spatial mapping highlighted region-specific expression and pathway activity within the seminiferous epithelium. Immunofluorescence staining validated the cell type–specific localization of key markers at the protein level.

Conclusion: Our integrative analysis reveals how transcriptional, epigenetic, and spatial programs converge on a core regulatory network to orchestrate human spermatogenesis. These findings provide new insights into germ cell differentiation and establish a comprehensive framework for investigating the molecular basis of male infertility and testicular dysfunction.

Keywords: single-cell RNA sequencing, spermatids, integrative analysis, differentiation, transcription factors