MicroRNAs in neural stem cell proliferation and differentiation

Abstract

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that are emerging as key regulators of neural (stem) cell properties. Due to their ability to regulate a broad repertoire of targets, they represent an exciting tool to modulate stem cell fate. Brain-enriched miR-9/9* has been described to enhance neural stem<br /> cell differentiation and impact on regionalization. Furthermore, it was suggested to interact with the Notch signaling pathway, which is well known to play a fundamental role in neural stem cell maintenance and differentiation. <br /> To investigate a potential miRNA-Notch-interplay, a Notch-dependent population of neuroepithelial stem cells derived from human embryonic stem (lt-NES) cells was chosen as a model system. In line with previous findings, ectopic expression of miR-9/9* impaired self-renewal and promoted differentiation of lt-NES cells. Our analysis<br /> revealed that this impact of miR-9/9* is, at least in part, due to a feedback loop between miR-9/9* and Notch activity. Notch directly controls miR-9/9* expression on<br /> a transcriptional level, while miR-9/9* in turn regulates Notch signaling by targeting of HES1 and NOTCH2. However, elevated levels of individual miR-9 and miR-9*<br /> revealed a separate way of action. While miR-9 enhanced differentiation and targeted HES1 and NOTCH2, miR-9* enhanced differentiation, impaired self-renewal of lt-NES cells and was shown to target NOTCH2 and SOX2.<br /> Compared to other miRNAs identified in a miRNA expression profiling during lt-NES cell differentiation (i.e. miR-7, miR-128, and miR-130b), only elevated levels of miR-9/9* induced significant changes in lt-NES proliferation and spontaneous differentiation. Nevertheless, long-term (15 days) lt-NES cell differentiation was enhanced by miR-9/9* as well as miR-7. Detailed analysis revealed that they even affect neuronal subtype specification. While the overall number of differentiated neurons increased, the generation of neurons positive for dopaminergic marker TH was impaired upon miR-9/9* and miR-7 overexpression. In line with this, miR-9, miR-9*, and miR-7 were downregulated during the time course of a floor plate precursor based differentiation protocol for the generation of mesencephalic dopaminergic neurons from human pluripotent stem cells. Futhermore, overexpression of miR-9/9* and miR-7 reduced mRNA expression levels of EN2 and FOXA2, two additional dopaminergic markers.<br /> Taken together, these results show that miRNAs can be used to modulate human neural stem cell maintenance, differentiation and subtype specification. Specifically,<br /> miR-9 and miR-9* act on human neural stem cell maintenance and spontaneous differentiation – to some extent by their interplay with the Notch signaling pathway. In addition, miR-9/9* as well as miR-7 impact on neural stem cell differentiation and lineage decision.