mirVana™ Reagents and Kits
MicroRNAs and Neural Stem Cell Differentiation

MicroRNAs (miRNAs) are key regulators of gene expression and have been implicated in a variety of critical biological processes, including development, cell differentiation, and oncogenesis. To study this important class of small regulatory RNAs (19–23 nt in length), innovative and specialized tools are required. The mirVana™ kits and reagents have been developed to provide optimized solutions for miRNA isolation, expression profiling, quantitative detection, and functional studies. This article describes how Ambion’s complete suite of tools was used to study the involvement of miRNA in neural stem cell differentiation.

Yann Thomas, Sylvain Lehmann, and Ollivier Milhavet

Human Genetics Institute of Montpellier, Biology of Transmissible Spongiform Encephalopathies Unit, 141 Cardonille Street, 34396 MONTPELLIER Cedex 5, FRANCE

Figure 1. Differential miRNA Expression During Neuronal Differentiation. (A) Total RNA was isolated with the mirVana™ miRNA Isolation Kit from cultured mouse neural stem cells 0, 1, or 6 days (D0, D1, D6) after induction of neural progenitor differentiation. miRNA expression was assessed by Northern blot (10 µg total RNA) or by end point PCR (25 ng total RNA, 25 cycles) using the mirVana qRT-PCR miRNA Detection Kit and appropriate Primer Sets. 5S rRNA was used as a loading control. (B) The relative change in miRNA expression at day 1 and 6 was graphed according to the signal intensity at day 0 for the three independent detection methods. Microarray expression data were obtained after miRNA isolation with the flashPAGE™ Fractionator, labelling of the miRNA fractions with the mirVana miRNA Labelling Kit, and hybridization on microarrays custom printed using the mirVana miRNA Probe Set.

Model for Neural Development
Cultured stem cells from the central nervous system (CNS) have proved useful in defining the pathways that lead to generation of neurons and glia [1]. In culture, stem cells self-renew, and after mitogen withdrawal, differentiate into neurons, astrocytes, and oligodendrocytes in predictable proportions [1, 2]. Ex vivo, single extrinsic factors can shift the fate of CNS stem cells toward specific cell lineages [2, 3]. Based on these characteristics, these cells have received increasing attention because of the potential for their use in cell replacement or gene therapy.

Dr. Ollivier Milhavet and colleagues were interested in determining whether miRNA expression is regulated during neural stem cell differentiation and whether miRNA could be directly involved in the differentiation process. To establish a model system to study these questions, cortices were isolated from mouse embryos (embryonic day 13.5) to create a monolayer culture of neural stem cells [4]. Human recombinant basic fibroblast growth factor (bFGF; 25 ng/ml) was added daily to stimulate expansion of the proliferative precursors, and differentiation of the neural progenitors was induced by withdrawing bFGF treatment.

miRNA Profiling of Differentiating Neural Stem Cells
To identify potential variation of miRNA expression levels during differentiation, expression profiles were determined with miRNA arrays prepared from the mirVana miRNA Probe Set [5]. Total RNA was isolated from cortical cultures after 0, 1, or 6 days of differentiation, and following fractionation with the flashPAGE™ System, the population of mature miRNAs at day 0 was labeled using the mirVana miRNA Labeling Kit and compared to labeled miRNA populations at days 1 or 6. Of the 161 miRNA probes on the microarray, 39 miRNAs showed significant (at least 1.5 fold) differences in expression during neuronal differentiation. Seven of the 39 miRNAs were chosen for further analysis (manuscript in preparation).

Interestingly, different classes of miRNA were identified according to tightly regulated expression patterns. Several miRNAs showed steadily increasing or decreasing expression levels throughout differentiation. Expression levels of others were strongly up- or down-regulated at day 1, which by day 6 either remained unchanged or returned to levels similar to day 0 (data not shown). These observations suggest that different miRNAs may be involved in specific cellular processes such as exit of cell cycle, neural differentiation, or maintenance of the differentiated state.

Figure 2. Effect of Pre-miR™ and Anti-miR™ Molecules on Mouse Neuronal Differentiation. Pregnant female CD1 mice were sacrificed and cortices were dissected from embryos at day 13.5. Proliferative precursors were cultured for two days in the presence of bFGF and then electroporated with 5 µg of a specific Pre-miR miRNA Molecule or Anti-miR miRNA Inhibitor using Nucleofector® Technology (amaxa® Biosystems). bFGF was withdrawn from the culture medium to induce differentiation and cells were further grown for 6 days. Cell nuclei were stained with DAPI and differentiated neuronal cells were revealed by immunofluorescence with antibodies specific for bIII tubulin (Covance Research Products). As a control, nontransfected differentiated cells were also analyzed. A schematic representation of the observed neuronal bodies and dendrite extensions is shown under each picture. (See Figure 1 for expression pattern of this miRNA during differentiation.)

Profiling data were also confirmed by two independent detection methods. An example of these validation tests for a specific miRNA that showed a steady increase of expression throughout differentiation according to the microarray analysis is shown in Figure 1A. A direct comparison of the results showed very good correlation among the three detection methods used (Figure 1B). As expected, the expression level of this miRNA, relative to the internal control 5S rRNA, significantly increased at day 1 and at day 6 by Northern blot analysis and qRT-PCR (mirVana qRT-PCR miRNA Detection Kit and Primer Sets).

Functional Analysis of Differentially Expressed miRNAs
To further characterize these miRNAs, their expression levels in neural stem cells were either augmented by electroporation of Pre-miR™ miRNA Precursor Molecules or blocked by electroporation of the corresponding Anti-miR™ miRNA Inhibitors. Cells were then allowed to grow and differentiate after bFGF withdrawal for 6 additional days, and the impact on differentiation was assayed by immunofluorescence experiments using specific cell markers (e.g., neuronal marker bIII tubulin). When targeting an miRNA that showed a strong increase in expression during differentiation (see Figure 1), no differences were observed in the number of neurons produced after either Pre-miR or Anti-miR treatment (Figure 2). However, Pre-miR miRNA overexpression early during differentiation resulted in simplified branching structures, and inhibition by Anti-miR electroporation resulted in longer neuronal extensions compared to control cells. These experiments confirm that alteration of the tightly regulated expression levels of this miRNA affects the outcome of neural progenitor differentiation.

Conclusions
In summary, these results show that miRNAs are directly implicated in the differentiation of mouse cortical neural stem cells ex vivo. The use of optimized miRNA research tools allowed accurate expression profiling and rapid functional validation of specific miRNAs. The remaining challenge to determining the precise function of miRNA is to discover their cellular targets. Dr. Milhavet and colleagues are currently performing experiments to identify potential messenger RNAs regulated by the miRNAs identified in this study.

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