Network-like Impact of MicroRNAs on Neuronal Lineage Differentiation of Unrestricted Somatic Stem Cells from Human Cord Blood (USSC)

Unrestricted somatic stem cells (USSC) represent an intrinsically multipotent CD45-negative population from human cord blood. They show differentiation into neuronal cells of a dopaminergic phenotype which express neuronal markers like synaptophysin, neuronal-specific nuclear protein (NeuN), and neurofilament and release the neurotransmitter dopamine accompanied by expression of dopaminergic key factors tyrosine hydroxylase and Nurr1 (NR4A2). MicroRNA expression analysis revealed downregulation of a set of 18 microRNAs during neuronal lineage differentiation of USSC, including members of the miR-17-92 family and additional microRNAs like miR-130a, -138, -218, and -335. In-silico target gene predictions for this microRNA group uncovered a large set of proteins involved in neuronal differentiation a strong impact on differentiation-related pathways like Axon Guidance, TGFss-, WNT-, and MAPK-Signaling. Experimental target validations confirmed approximately 35% of predictions tested and revealed a group of proteins with specific impact in neuronal differentiation and function including neurobeachin (NBEA), neurogenic differentiation 1 (NEUROD1), cysteine-rich motor neuron protein 1 (CRIM1), neuropentraxin 1 (NPTX1), and others. These proteins are combined targets for several subgroups from the set of 18 downregulated microRNAs. This finding was further supported by the observed upregulation of a significant amount of predicted and validated target genes based on Illumina Beadstudio microarray data. Confirming the functional relationship of a limited panel of microRNAs and predicted target proteins reveals a clear network-like impact of the group of 18 downregulated microRNAs on proteins with functions involved in neuronal development and function