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Abstract
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Spermatogonial stem cells (SSCs) are unipotent germline cells with emerging pluripotent
potential under specific in vitro conditions. Understanding their capacity for reprogramming
and the molecular mechanisms involved offers valuable insights into regenerative
medicine and fertility preservation. SSCs were isolated from Oct4-GFP C57BL/6 transgenic
mice using enzymatic digestion and cultured in defined media. Under these conditions,
ES-like colonies emerged expressing pluripotency markers. These cells were characterized
by immunocytochemistry, teratoma assays, and transcriptomic analyses using bulk and
single-cell RNA sequencing datasets. Gene expression profiles were compared with ESCs
and SSCs using datasets from GEO (GSE43850, GSE38776, GSE149512). Protein–protein
interaction (PPI) networks and co-expression modules were explored through STRING,
Cytoscape, and WGCNA. ES-like cells derived from SSCs exhibited strong expression of
OCT4, DAZL, and VASA. Transcriptomic analysis revealed key differentially expressed
genes and shared regulatory networks with ESCs. WGCNA identified key co-expression
modules and hub regulatory RNA binding genes (Ctdsp1, Rest, and Stra8) potentially
responsible for the reprogramming process. Teratoma assays confirmed pluripotency,
and single-cell RNA-seq validated expression of critical markers in cultured SSCs. This
study demonstrates that SSCs can acquire pluripotency features and be reprogrammed into
ES-like cells. The integration of transcriptomic and network-based analyses reveals novel
insights into the molecular drivers of SSC reprogramming, highlighting their potential
utility in stem cell-based therapies and male fertility preservation.
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