Characterization of novel microRNAs associated with differentiation of human embryonic stem cell-derived neural stem cells

MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional regulators of gene expression and influence many diverse biological processes including the development and function of the nervous system. Although studies in model organisms have identified several microRNAs that critically impact on neuronal differentiation, data on microRNA functions in the context of human neural development and neural stem cell biology are still scarce. In an attempt to fill this gap, we assessed microRNA expression profiles of human embryonic stem cells (hESC, I3 line), of derived long term self-renewing neural stem cells (hESNSC) and of further differentiated neuronal cultures (ND15, ND30) by using a quantitative real-time PCR multiplex assay. Our analysis shows that microRNA expression patterns change dramatically during neural differentiation. We validated the observed changes in microRNA expression by Northern blot analysis in two different hES cell lines (I3 and H9.2) indicating the reliability of our approach. In addition, we were able to confirm previous assignments of ES cell-specific and neuronal-specific microRNAs. We have started to assess the function of several novel microRNAs that were identified as up-regulated during neuronal differentiation and not yet studied in the context of human neural development by performing gain of function experiments in NSCs using a lentiviral-based transduction method. Stable over-expression of these newly identified microRNAs, just like the over-expression of known neurogenesis-associated microRNAs, i.e. miR-124 and miR125, resulted in an increased rate of neuronal differentiation. Our ES cell-based neural differentiation paradigm should provide a useful tool for the study of microRNA signatures in human pluripotent and multipotent stem cells and for the identification and functional characterization of novel microRNAs associated with human neural development.