Exosome-Mediated Ultra-Effective Direct Conversion of Human Fibroblasts into Neural Progenitor-like Cells.

Exosomes, naturally secreted nanoparticles, have been introduced as vehicles for horizontal transfer of genetic material. We induced autologous exosomes containing a cocktail of reprogramming factors ("reprosomes") to convert fibroblasts into neural progenitor cells (NPCs). The fibroblasts were treated with ultrasound and subsequently cultured in neural stem cell medium for 1 day to induce the release of reprosomes composed of reprogramming factors associated with chromatin remodeling and neural lineage-specific factors. After being treated with reprosomes, fibroblasts were converted into NPCs (rNPCs) with great efficiency via activation of chromatin remodeling, so quickly that only 5 days were required for the formation of 1500 spheroids showing an NPC-like phenotype. The rNPCs maintained self-renewal and proliferative properties for several weeks and successfully differentiated into neurons, astrocytes, and oligodendrocytes in vitro and in vivo. Reprosome-mediated cellular reprogramming is simple, safe, and efficient to produce autologous stem cells for clinical application.

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 multiple piRNAs in a single breast cancer cell using molecular beacons.

Simultaneous bioimaging of piR-36026 and piR-36743 using molecular beacons successfully visualized 4 different subtypes of breast cancer.

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.

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 cells in vitro and in 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 both in vitro and in vivo in G2 cells induced to differentiate into neurons. However, in vitro and in 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.

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.

A self-assembling magnetic resonance beacon for the detection of microRNA-1.

A self-assembling magnetic resonance beacon was used to visualize the microRNA-1 expression-dependent change in magnetic resonance signal intensity.

Bioimaging of the microRNA-294 expression-dependent color change in cells by a dual fluorophore-based molecular beacon.

A dual fluorophore-based color-tunable molecular beacon visualized the microRNA-294 expression-dependent color change in cells.

Magnetic resonance beacon to detect intracellular microRNA during neurogenesis.

Magnetic resonance imaging (MRI) offers great spatial resolution for viewing deep tissues and anatomy. We developed a self-assembling signal-on magnetic fluorescence nanoparticle to visualize intracellular microRNAs (miRNAs or miRs) during neurogenesis using MRI. The self-assembling nanoparticle (miR124a MR beacon) was aggregated by the incubation of three different oligonucleotides: a 3' adaptor, a 5' adaptor, and a linker containing miR124a-binding sequences. The T2-weighted magnetic resonance (MR) signal of the self-assembled nanoparticle was quenched when miR124a was absent from test tubes or was minimally expressed in cells and tissues. When miR124a was present in test tubes or highly expressed in vitro and in vivo during P19 cell neurogenesis, it hybridized with the miR124a MR beacon, causing the linker to detach, resulting in increased signal-on MRI intensity. This MR beacon can be used as a new imaging probe to monitor the miRNA-mediated regulation of cellular processes.

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.

Evaluation of antiangiogenic effects of a new synthetic candidate drug KR-31831 on xenografted ovarian carcinoma using dynamic contrast enhanced MRI.

OBJECTIVE: The purpose of this research was to investigate the anti-angiogenic inhibitory effect of KR-31831, a newly developed anti-angiogenic agent, on an in vivo human ovarian carcinoma model using dynamic contrast-enhanced (DCE) MRI. MATERIALS AND METHODS: Xenografted ovarian tumors were established by subcutaneous injection of SKOV3 cells into mice. The mice were treated daily with KR-31831 at 50 mg/kg for 21 days. Tumor tissues were excised corresponding to the DCE-MRI sections for evaluation of MVD with CD31 immunohistochemistry. All in vivo MRIs were performed on a 7.0 Tesla micro-MRI System. DCE-MRI was acquired prior to initiating treatment with KR-31831 and again on days 3 and 21 after treatment. The permeability parameters (K(trans), v(e), and v(p)) were estimated using a pharmacokinetic model. RESULTS: Qualitatively, the K(trans) parametric mapping showed different changes before and after treatment with KR-31831 in the treatment group. For quantification of this change, the median of K(trans) values were compared before and after treatments in the control and KR-31831-treated groups. A non-parametric statistical test (Wilcoxon signed-rank test) showed decreasing K(trans) values on day 21 compared to days 0 and 3 in the KR-31831-treated group (p < 0.05), whereas there was no significant difference in the control group (p = 0.84). CONCLUSION: Our results suggest that DCE-MRI can be a useful tool by which to evaluate the anti-angiogenic effect of KR-31831 on a xenografted human ovarian carcinoma model.

Development of target-specific multimodality imaging agent by using hollow manganese oxide nanoparticles as a platform.

A platform protocol developed based on the hollow manganese oxide nanoparticles provided multimodal diagnostic agents, which allow the selectively detect vulva cancer with T(1)-weighted in vivo MRI.

pH-responsive polymeric micelle based on PEG-poly(ß-amino ester)/(amido amine) as intelligent vehicle for magnetic resonance imaging in detection of cerebral ischemic area.

A series of pH-responsive polymeric micelles is developed to act as intelligent carriers to deliver iron oxide (Fe(3)O(4)) nanoparticles and respond rapidly to an acidic stimuli environment for magnetic resonance imaging (MRI). The polymeric micelle can be self-assembled at physiological pH by a block copolymer, consisting of a hydrophilic methoxy poly(ethylene glycol) (PEG) and a pH-responsive poly(ß-amino ester)/(amido amine) block. Consequently, the Fe(3)O(4) nanoparticles can be well encapsulated into polymeric micelles due to the hydrophobic interaction, shielded by a PEG coronal shell. In an acidic environment, however, the pH-responsive component, which has ionizable tert-amino groups on its backbone, can become protonated to be soluble and release the hydrophobic Fe(3)O(4) nanoparticles. The Fe(3)O(4)-loaded polymeric micelle was measured by dynamic light scattering (DLS), superconducting quantum interference device (SQUID) and a 3.0T MRI scanner. To assess the ability of this MRI probe as a pH-triggered agent, we utilize a disease rat model of cerebral ischemia that produces acidic tissue due to its pathologic condition. We found gradual accumulation of Fe(3)O(4) nanoparticles in the brain ischemic area, indicating that the pH-triggered MRI probe may be effective for targeting the acidic environment and diagnostic imaging of pathologic tissue.

Fabrication of a silica sphere with fluorescent and MR contrasting GdPO4 nanoparticles from layered gadolinium hydroxide.

The delaminated gadolinium hydroxide layers doped with Eu(3+) ions were assembled on the surface of silica spheres and annealed at high temperatures, resulting in the formation of fluorescent and MR active GdPO(4) : Eu nanoparticles at the surface.

Apoptosis signal-regulating kinase 1 regulates the expression of caspase-11.

Caspase-11 is an inducible caspase involved in the regulation of cell death and inflammation. In the present study, we examined whether apoptosis signal-regulating kinase 1 (Ask1)-mediated signaling pathway is involved in the expression of caspase-11 induced by lipopolysaccharide (LPS). We found that the induction of caspase-11 was suppressed by the inhibitors of NADPH oxidase (Nox) or knockdown of Nox4 that acts downstream of toll-like receptor 4 and generates Ask1-activating reactive oxygen species. Overexpression of dominant negative tumor necrosis factor receptor associate factor 6 also suppressed the induction of caspase-11. Importantly, knockdown or dominant negative form of Ask1 suppressed the induction of caspase-11 following LPS stimulation. Taken together, our results show that Ask1 regulates the expression of caspase-11 following LPS stimulation.

Suppression of caspase-11 expression by histone deacetylase inhibitors.

It has been well documented that histone deacetylase inhibitors suppress inflammatory gene expression. Therefore, we investigated whether histone deacetylase inhibitors modulate the expression of caspase-11 that is known as an inducible caspase regulating both inflammation and apoptosis. In the present study, we show that sodium butyrate and trichostatin A, two structurally unrelated inhibitors of histone deacetylase (HDAC), effectively suppressed the induction of caspase-11 in mouse embryonic fibroblasts stimulated with lipopolysaccharides. Sodium butyrate inhibited the activation of upstream signaling events for the caspase-11 induction such as activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, degradation of inhibitor of kappaB, and activation of nuclear factor-kappaB. These results suggest that the HDAC inhibitor suppressed cytosolic signaling events for the induction of caspase-11 by inhibiting the deacetylation of non-histone proteins.