Functional evaluation of microrna-networks of neuronal differentiation in cell models of limited and full differentiation capability

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MicroRNAs as important posttranscriptional regulators are essentially involved in the regulation of many biological processes. They play pivotal roles in the complex but yet poorly understood networks of molecular players controlling neuronal differentiation which involve precise coordination in the time course and level of expression. For their experimental interrogation stem cells have emerged as a valuable and promising tool.Unrestricted somatic stem cells from human cord blood (USSC) can be induced to limited neuronal differentiation by a cocktail of growth- and differentiation factors (XXL-USSC). In addition, they can be reprogrammed to induced pluripotent stem cells (ipUSSC) of full differentiation potential by ectopic retroviral expression of OCT4, SOX2, KLF4, and C-MYC. Here we aim to compare and functionally evaluate the microRNA-regulated molecular networks controlling neuronal differentiation of USSC and ipUSSC.We hypothesize that the limitations in neuronal differentiation of USSC are due to deregulated checkpoints, a not fully functional network of molecular players which can be revealed by comparison with the full neuronal differentiation observed with ipUSSC. Upon identification of interacting molecular players, a targeted experimental intervention should reveal a general signature of checkpoints relevant for neuronal differentiation.In the first step of our proposed approach, we will generate a comprehensive picture of mRNA, microRNA and protein in partial and complete neuronal differentiation of USSC and ipUSSC, in order to obtain an overview of coordinated gene expression, posttranscriptional regulation and posttranslational processes during neuronal differentiation. Recently, we identified 18 microRNAs, mainly members of the miR-17-92 family, that are downregulated in XXL-USSC and experimentally validated numerous bioinformatically predicted target genes important in neuronal differentiation and related biochemical pathways. Based on the expression pictures, the central part of our study aims to identify deregulated checkpoints in USSC by substantial bioinformatic analysis followed by functional validation of microRNA candidates. The latter will be achieved by experimental validation of candidate microRNA target genes followed by perturbation analysis using mixtures of microRNA-mimics, -inhibitors and siRNAs followed by induction to differentiation. Rather than solely focusing on single factors, mixes of small RNAs allow to more closely mimic patterns of real expression changes of several candidates. Morphological and molecular neuro-specific read-outs, supported by proteome analysis will allow us to test the functional input of candidates. These analyses will provide new functional insights into microRNAs in molecular networks and checkpoints of neuronal differentiation and define starting points for further investigations within the priority programme.