Abstract
BackgroundSpermatogonial stem cells (SSCs) have the unique ability to undergo self-renewal division. However, these cells are morphologically indistinguishable from committed spermatogonia, which have limited mitotic activity. To establish a system for SSC purification, we analyzed the expression of SSC markers CD9 and epithelial cell adhesion molecule (EPCAM), both of which are also expressed on embryonic stem (ES) cells. We examined the correlation between their expression patterns and SSC activities.Methodology and Principal FindingsBy magnetic cell sorting, we found that EPCAM-selected mouse germ cells have limited clonogenic potential in vitro. Moreover, these cells showed stronger expression of progenitor markers than CD9-selected cells, which are significantly more enriched in SSCs. Fluorescence-activated cell sorting of CD9-selected cells indicated a significantly higher frequency of SSCs among the CD9+EPCAMlow/- population than among the CD9+EPCAM+ population. Overexpression of the active form of EPCAM in germline stem (GS) cell cultures did not significantly influence SSC activity, whereas EPCAM suppression by short hairpin RNA compromised GS cell proliferation and increased the concentration of SSCs, as revealed by germ cell transplantation.Conclusions/SignificanceThese results show that SSCs are the most concentrated in CD9+EPCAMlow/- population and also suggest that EPCAM plays an important role in progenitor cell amplification in the mouse spermatogenic system. The establishment of a method to distinguish progenitor spermatogonia from SSCs will be useful for developing an improved purification strategy for SSCs from testis cells.
Highlights
Spermatogonial stem cells (SSCs) account for a small population of testis cells [1,2], and their self-renewal activity distinguishes them from committed progenitor cells
Conclusions/Significance: These results show that SSCs are the most concentrated in CD9+EPCAMlow/- population and suggest that epithelial cell adhesion molecule (EPCAM) plays an important role in progenitor cell amplification in the mouse spermatogenic system
Testis cells were prepared from 7-day-old pups, and EPCAM-expressing cells were collected by magnetic cell sorting (MACS)
Summary
Spermatogonial stem cells (SSCs) account for a small population of testis cells [1,2], and their self-renewal activity distinguishes them from committed progenitor cells. In 1994, a germ cell transplantation technique was developed, in which donor testis cells recolonize seminiferous tubules following microinjection into the testes of infertile recipients [3] This provided the first functional assay for SSCs. The estimated number of SSCs was 26103 to 36103 per testis, which represents ,10% of the total Asingle (As) spermatogonia, suggesting that only a small population of As cells have SSC activity [2,4,5]. Spermatogonial stem cells (SSCs) have the unique ability to undergo self-renewal division These cells are morphologically indistinguishable from committed spermatogonia, which have limited mitotic activity. We examined the correlation between their expression patterns and SSC activities
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