MicroRNA Expression Profiling of Multipotent Adult Germline Stem Cells

Spermatogonial stem cells (SSCs) isolated from the adult mouse testis and cultured have been shown to respond to culture conditions and become pluripotent, so called multipotent adult germline stem cells (maGSCs). Recently a new group of non coding RNAs, called microRNAs (miRNAs), has been implicated to play a key role in regulation of embryonic development and pluripotency in mouse embryonic stem cells (ESCs). The objective of the current study was to test similarities and differences between maGSCs and ESCs with respect to miRNAs. Whole miRNA array expression profiling showed that despite slight differences in expression of some oncomirs, undifferentiated maGSCs and ESCs originating from 129/Sv mouse strain are equivalent cell types from the viewpoint of microRNAome. In contrast, undifferentiated maGSCs originating from the transgenic Stra8-EGFP-Rosa mouse are a distinct cell type regarding miRNAs. miRNAs belonging to the 290 and 302 clusters have been previously classified as ESC-specific. This study shows that these miRNAs generally characterize pluripotent cells. They are expressed not only in undifferentiated ESCs but also in undifferentiated maGSCs as well as in the F9 embryonic carcinoma cell (ECC) line. In addition, this study tested the time-dependent influence of different factors that promote loss of pluripotency on levels of these miRNAs in all three pluripotent cell types. Using standard methods to induce ESC differentiation, this study showed loss of stem cell markers and miR-290 family expression in ESCs. Likewise, maGSC cultures lost pluripotent marker expression and miR-290 family expression was strongly attenuated, albeit slower than in ESCs. In addition, ESC differentiation with gelatin led to a marked and early upregulation of the miR-302 family that was lost over time, while a slow and steady upregulation was observed in maGSCs treated similarly. Differences in time and extent of differentiation observed between ESCs and maGSCs correspond to similar differences in the expression profiles of both miRNA families, suggesting similar underlying mechanisms in maintenance of pluripotency between the two cell types. These results indicate that the 290-miRNA family is connected with Oct-4 and maintenance of the pluripotent state. In contrast, members of the 302-miRNA family are induced during first stages of in vitro differentiation in all cell types tested. Therefore, detection of miRNAs of miR-302 family in pluripotent cells could be attributed to the proportion of spontaneously differentiating cells in cultures of pluripotent cells. Furthermore, differences in the miRNA levels observed between ESCs and maGSCs correlate with differences observed in expression of de novo DNA methyltransferases and global methylation state in these cells. All these results are consistent with ESC-like nature of maGSCs and their potential as an alternative source of pluripotent cells from non-embryonic tissues. At the same time, however, they imply that the mechanisms that control differentiation in pluripotent cells may differ in some aspects between ESCs and maGSCs, and miRNAs may be responsible for these differences.