Identification of an intermediate state as spermatogonial stem cells reprogram to multipotent cells.
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 morphologically distinct intermediate state with characteristic expression patterns of pluripotency-related genes and miRNAs 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.
Kim
Hyung Joon
HJ
Fertility Center of CHA Gangnam Medical Center, College of Medicine, CHA University, Seoul, 135-081, Korea.
Lee
Hyun Jung
HJ
Lim
Jung Jin
JJ
Kwak
Ki Hoon
KH
Kim
Jong Soo
JS
Kim
Ji Hoon
JH
Han
Yong-Mahn
YM
Kim
Kye-Seong
KS
Lee
Dong Ryul
DR
D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation; D049109:Cell Proliferation; D054338:Cell Transdifferentiation / Q000235:genetics; D002478:Cells, Cultured; D018920:Coculture Techniques; D019175:DNA Methylation; D005347:Fibroblasts / Q000473:pathology; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D035683:MicroRNAs / Q000032:analysis*; D039902:Multipotent Stem Cells / Q000378:metabolism* / Q000473:pathology; D013091:Spermatogenesis / Q000235:genetics; D013093:Spermatogonia / Q000378:metabolism* / Q000473:pathology
D035683:MicroRNAs