Search

Osteoblast differentiation and bone formation (osteogenesis) are regulated by transcriptional and post‐transcriptional mechanisms. Recently, microRNAs (miRNAs) were identified as novel key regulators of human stromal (skeletal, mesenchymal) stem cells (hMSC) differentiation. Here, we identified miRNA‐34a (miR‐34a) and its target protein networks as modulator of osteoblastic (OB) differentiation of hMSC. miRNA array profiling and further validation by quantitative RT‐PCR revealed that miR‐34a was upregulated during OB differentiation of hMSC, and in situ hybridization confirmed its OB expression in vivo. Overexpression of miR‐34a inhibited early commitment and late OB differentiation of hMSC in vitro, whereas inhibition of miR‐34a by anti‐miR‐34a enhanced these processes. Target prediction analysis and experimental validation confirmed Jagged1 (JAG1), a ligand for Notch 1, as a bona fide target of miR‐34a. siRNA‐mediated reduction of JAG1 expression inhibited OB differentiation. Moreover, a number of known cell cycle regulator and cell proliferation proteins, such as cyclin D1, cyclin‐dependent kinase 4 and 6 (CDK4 and CDK6), E2F transcription factor three, and cell division cycle 25 homolog A were among miR‐34a targets. Furthermore, in a preclinical model of in vivo bone formation, overexpression of miR‐34a in hMSC reduced heterotopic bone formation by 60%, and conversely, in vivo bone formation was increased by 200% in miR‐34a‐deficient hMSC. miRNA‐34a exhibited unique dual regulatory effects controlling both hMSC proliferation and OB differentiation. Tissue‐specific inhibition of miR‐34a might be a potential novel therapeutic strategy for enhancing in vivo bone formation. StemCells2014;32:902–912

A microfluidic component library for building systems driving parallel or serial microfluidic-based assays is presented. The components are a miniaturized eight-channel peristaltic pump, an eight-channel valve, sample-to-waste liquid management, and interconnections. The library of components was tested by constructing various systems supporting perfusion cell culture, automated DNA hybridizations, and in situ hybridizations. The results showed that the MainSTREAMcomponents provided (1) a rapid, robust, and simple method to establish numerous fluidic inputs and outputs to various types of reaction chips; (2) highly parallel pumping and routing/valving capability; (3) methods to interface pumps and chip-to-liquid management systems; (4) means to construct a portable system; (5) reconfigurability/flexibility in system design; (6) means to interface to microscopes; and (7) compatibility with tested biological methods. It was found that LEGO Mindstorms motors, controllers, and software were robust, inexpensive, and an accessible choice as compared with corresponding custom-made actuators. MainSTREAMsystems could operate continuously for weeks without leaks, contamination, or system failures. In conclusion, the MainSTREAMcomponents described here meet many of the demands on components for constructing and using microfluidics systems.

The use of short, high-affinity probes consisting of a combination of DNA and locked nucleic acid (LNA) has enabled the specific detection of microRNAs (miRNAs) by in situ hybridization (ISH). However, detection of low–copy number miRNAs is still not always possible. Here the authors show that probes consisting of 2′-O-methyl RNAs (2OMe) and LNA at every third base (2:1 ratio), under optimized hybridization conditions, excluding yeast RNA from the hybridization buffer, can provide superior performance in detection of miRNA targets in terms of sensitivity and signal-to-noise ratio compared to DNA + LNA probes. Furthermore, they show that hybridizations can be performed in buffers of 4M urea instead of 50% formamide, thereby yielding an equally specific but nontoxic assay. The use of 2OMe + LNA–based probes and the optimized ISH assay enable simple and fast detection of low–copy number miRNA targets, such as miR-130a in mouse brain.