Hyejung Heo

A color-tunable molecular beacon to sense miRNA-9 expression during neurogenesis

A typical molecular beacon (MB) composing of a fluorophore and a quencher has been used to sense various intracellular biomolecules including microRNAs (miRNA, miR). However, the on/off-tunable miRNA MB is difficult to distinguish whether the observed low fluorescence brightness results from low miRNA expression or low transfection of the miRNA MB. We developed a color-tunable miRNA-9 MB (ColoR9 MB) to sense miR-9 expression-dependent color change. The ColoR9 MB was synthesized by a partially double-stranded DNA oligonucleotide containing a miR-9 binding site and a reporter probe with Cy3/black hole quencher 1 (BHQ1) at one end and a reference probe with Cy5.5 at the other end. The ColoR9 MB visualized CHO and P19 cells with red color in the absence of miR-9 and yellow color in the presence of miR-9. In vivo imaging demonstrated that the green fluorescence recovery of the reporter probe from the ColoR9 MB increased gradually during neuronal differentiation of P19 cells, whereas red fluorescence activity of the reference probe remained constant. These results showed the great specificity of sensing miR-9 expression- and neurogenesis-dependent color change.

Multimodal imaging probe for targeting cancer cells using uMUC-1 aptamer

For adequate cancer therapy, newer imaging modalities with more specific ligands for unique targets are crucial. Underglycosylated mucin-1 (uMUC-1) antigen is an early marker of tumor development and is widely overexpressed on most tumors. A combination of nanotechnology with optical, radionuclide, and magnetic resonance (MR) imaging has great potential to improve cancer diagnosis and therapy. In this study, a multimodal nanoparticle imaging system was developed that can be used for optical, MR and positron emission tomography (PET) imaging. Cobalt ferrite magnetic nanoparticles surrounded by fluorescent rhodamine (designated MF) within a silica shell matrix were conjugated with an aptamer targeting uMUC-1 (designated MF-uMUC-1) and further labeled by (68)Ga (designated MFR-uMUC-1) with the help of a p-SCN-bn-NOTA chelating agent, resulting in single multimodal nanoparticles. The resultant nanoparticles are spherical and monodispersed, as revealed by transmission electron microscopy. The MFR-uMUC-1 nanoparticle showed specific and dose-dependent fluorescent, radioisotope and MR signals targeting BT-20 cells expressing uMUC-1. In vivo targeting and multimodal imaging in tumor-bearing nude mice also showed great specificity for targeting cancers with MFR-uMUC-1. The MFR-uMUC-1 probe could be used as a single multimodal probe to visualize cancer cells by means of optical, radionuclide and MR imaging.

An ultra-effective method of generating extramultipotent cells from human fibroblasts by ultrasound.

Multipotent cells have similar basic features of all stem cells but limitation in ability of self-renewal and differentiation compared with pluripotent cells. Here, we have developed an ultra effective, gene- and chemical-free method of generating extra multipotent (xpotent) cells which have differentiation potential more than limited cell types, by the mechanism of ultrasound-directed permeation of environmental transition-guided cellular reprogramming (Entr). Ultrasound stimulus generated a massive number of Entr-mediated xpotent (x/Entr) spheroids from human dermal fibroblasts (HDFs) 6 days after treatment. The emergence of x/Entr was first initiated by the introduction of human embryonic stem cell (ESC) environments into the HDFs to start fast cellular reprogramming including activation of stress-related kinase signaling pathways, subsequent chromatin remodeling, and expression of pluripotent-related genes via transient membrane damage caused by ultrasound-induced cavitation. And then, pluripotent markers were transported into their adjacent HDFs via direct cell-to-cell connections in order to generate xpotent clusters. The features of x/Entr cells were intermediate between pluripotency and multipotency in terms of pluripotency with three germ layer markers, multi-lineage differentiation potential, and no teratoma formation. This physical stimulus-mediated reprogramming strategy was cost-effective, simple, quick, produced significant yields, and was safe, and can therefore provide a new paradigm for clinical application.

Bioimaging of microRNA124a-independent neuronal differentiation of human G2 neural stem cells.

Evaluation of the function of microRNAs (miRNAs or miRs) through miRNA expression profiles during neuronal differentiation plays a critical role not only in identifying unique miRNAs relevant to cellular development but also in understanding regulatory functions of the cell‐specific miRNAs in living organisms. Here, we examined the microarray‐based miRNA expression profiles of G2 cells (recently developed human neural stem cells) and monitored the expression pattern of known neuron‐specific miR‐9 and miR‐124a during neuronal differentiation of G2 cellsin vitro andin vivo. Of 500 miRNAs analyzed by microarray of G2 cells, the expression of 90 miRNAs was significantly increased during doxycycline‐dependent neuronal differentiation of G2 cells and about 60 miRNAs showed a gradual enhancement of gene expression as neuronal differentiation progressed. Real‐time PCR showed that expression of endogenous mature miR‐9 was continuously and gradually increased in a pattern dependent on the period of neuronal differentiation of G2 cells while the increased expression of neuron‐specific mature miR‐124a was barely observed during neurogenesis. Our recently developed miRNA reporter imaging vectors (CMV/Gluc/3×PT_miR‐9 and CMV/Gluc/3×PT_miR‐124a) containing Gaussia luciferase, CMV promoter and three copies of complementary nucleotides of each corresponding miRNA showed that luciferase activity from CMV/Gluc/3×PT_miR‐9 was gradually decreased bothin vitro andin vivo in G2 cells induced to differentiate into neurons. However,in vitro andin vivo bioluminescence signals for CMV/Gluc/3×PT_miR‐124a were not significantly different between undifferentiated and differentiated G2 cells. Our results demonstrate that biogenesis of neuron‐specific miR‐124a is not necessary for doxycycline‐dependent neurogenesis of G2 cells.

VisuFect-mediated siRNA delivery into zygotes

Current methods for delivering foreign genetic materials into mammalian cells are highly successful. However, these methods cannot be applied in oocyte or embryo systems due to their toxicity and low efficiency. Moreover, no satisfactory methods exist for delivering foreign genetic material without inducing physical damage to membranes. Here we developed an organic compound (VisuFect)-mediated small interfering RNA (siRNA) delivery method and evaluated this method in P19 cells and mouse zygotes. Oct4-siRNA conjugated VisuFect (Oct4-siRNA-VF) permeated the zona pellucida effectively and localized inside mouse zygotes without inducing membrane damage. Successful VisuFect-mediated delivery was further demonstrated by strong transcriptional repression of Oct4 expression by the delivered Oct4-siRNA, in addition to repressed embryonic development of mouse zygotes.

Corrigendum to "Magnetic resonance beacon to detect intracellular microRNA during neurogenesis" [Biomaterials 41 (2015) 69-78].

Corrigendum to “Magnetic resonance beacon to detect intracellular microRNA during neurogenesis” [Biomaterials 41 (2015) 69e78] Jonghwan Lee a, , Yeon A. Jin a, , Hae Young Ko a, , Yong Seung Lee a, , Hyejung Heo a, , Sujeong Cho a, , Soonhag Kim a, b, * a Institute for Bio-Medical Convergence, College of Medicine, Catholic Kwandong University, Gangneung-si, Gangwon-do 270-701, Republic of Korea b Catholic Kwandong University International St. Marys Hospital, Incheon Metropolitan City 404-834, Republic of Korea