The aim of this study was to understand the mechanisms that allow mSSC lines to be established from SSCs. Small, multilayer clumps of SSCs formed during two to four weeks of in vitro culture and were then transferred to MEF feeders. Small, round, monolayer colonies containing cells destined to convert to mSSCs, designated as intermediate state SSCs (iSSCs), first appeared after two to three passages. During an additional nine passages (47-54 days) under the same culture conditions, iSSCs slowly proliferated and maintained their morphology. Ultimately, a cell type with an ES-like morphology (mSSC) appeared from the iSSC colonies, and two mSSC cell lines were established. The mSSCs had a high proliferative potential in serum-free ES culture medium and have been successfully maintained since their first establishment (> 12 months). We also compared the specific characteristics of iSSCs with those of SSCs and mSSCs using immunocytochemistry, FACS, RT-PCR, DNA methylation, and miRNA analyses. The results suggest that iSSCs represent a morpholog-ically distinct intermediate state with characteristic ex-pression patterns of pluripotency-related genes and miR-NAs that arise during the conversion of SSCs into mSSCs. Our results suggest that iSSCs could be a useful model for evaluating and understanding the initiation mechanisms of cell reprogramming.

Keywords: culture-induced reprogramming, dedifferentiation, intermediate state, miRNA, spermatogonial stem cells