The impact of bifunctional microRNA-9/9* on the differentiation of human ES cell - derived neural stem cells

MicroRNAs are non-coding RNA molecules about 22-25 nucleotides long that regulate gene expression at a post-transcriptional level. MicroRNAs are known to be involved in many cellular processes including self-renewal and differentiation. We carried out a comparative microRNA expression profiling across the differentiation of human embryonic stem cells (hESC) into neural stem cells (hESNSC) and their neuronal progeny. MicroRNA-9/9* (miR-9/9*) was among the microRNAs that showed a strikingly differential expression pattern. It was almost absent in hESC, expressed in hESNSC and further up-regulated in differentiating neuronal cultures. Recent studies showed that miR-9 enhances neuronal differentiation of mouse neural progenitors and impacts on migration of human neural progenitors, while other studies showed that miR-9* antagonizes proliferation of mouse neural progenitors and contributes to neuronal development (Krichevsky, Sonntag et al. 2006; Yoo, Staahl et al. 2009; Delaloy, Liu et al. 2010). To gain insight into the potential roles of miR-9 and miR-9* in our long-term self-renewing hESNSC, we made use of a lentiviral-based over-expression system. By cloning the miR-9 locus under a suitable pol-III promoter we achieved over-expression of the mature forms of both miR-9 and miR-9* in hESNSC at comparable levels, which is ideal to study the role of bifunctional microRNAs. Despite the presence of growth factors (i.e. FGF2 and EGF) in the culture medium, miR-9/9* over-expression induced a reduction in proliferation and an increase in differentiation of hESNSC, as assessed by BrdU incorporation and expression of the neuronal marker beta-III-tubulin. By exposing hESNSC to individual synthetic mimics of miR-9 and miR-9*, we could further demonstrate that the promotion of differentiation is due to both miR-9 and miR-9*, whereas the anti-proliferative effect is mainly exerted by miR-9* alone. These findings are corroborated by target prediction analysis. Among the targets of miR-9* we found several cell cycle-associated genes, while among the targets of both miR-9 and miR-9* we found antagonists of differentiation.
The experimental approach presented here provides an elegant way to assess the role of microRNAs – including bifunctional ones such as miR-9/9* - during early human neural development, and offers the potential to modulate microRNA levels as attractive tool for gaining control on stem cell fate.