PSP1, a Phosphatidylserine-Recognizing Peptide, Is Useful for Visualizing Radiation-Induced Apoptosis in Colorectal Cancer In Vitro and In Vivo.

Accurate and timely visualization of apoptotic status in response to radiation is necessary for deciding whether to continue radiation or change to another mode of treatment. This is especially critical in patients with colorectal cancer, which requires a delicate combination of surgery, radiation, and chemotherapy in order to achieve optimal outcome. In this study, we investigated the potential of phosphatidylserine-recognizing peptide 1 (PSP1) as an apoptosis-targeting probe, which identifies phosphatidylserine on cell surfaces. We first screened colon cancer cell lines for their sensitivity to radiation and selected two cell lines: HCT116 and HT29. Cell binding assay using fluorescence-activated cell sorting and optical imaging showed that HCT116 cells had better binding to PSP1 than HT29 cells. Thus, mouse xenograft model using HCT116 cells was generated and was topically irradiated with either single or fractionated dose of radiation followed by systemic administration of PSP1 for subsequent molecular optical imaging. We confirmed that the PSP1 probe was selectively bound to apoptosis-induced tumor in a radiation dose-dependent manner. We also observed that fractionated radiation regimen, which is recently being used in clinical situation, was more effective in inducing tumor apoptosis than corresponding single-dose radiation treatment. We then evaluated the correlation between tumor targeting of PSP1 and suppression effect of tumor development and found that tumor volume and fluorescence intensity were correlated before (correlation coefficient r2 = 0.534) and after (r2 = 0.848) radiation therapy. Our study shows that PSP1 peptide is an efficient index probe for deciding "go or no-go" for radiation therapy in colorectal cancer.

Aucubin Promotes Differentiation of Neural Precursor Cells into GABAergic Neurons.

Aucubin is a small compound naturally found in traditional medicinal herbs with primarily anti-inflammatory and protective effects. In the nervous system, aucubin is reported to be neuroprotective by enhancing neuronal survival and inhibiting apoptotic cell death in cultures and disease models. Our previous data, however, suggest that aucubin facilitates neurite elongation in cultured hippocampal neurons and axonal regrowth in regenerating sciatic nerves. Here, we investigated whether aucubin facilitates the differentiation of neural precursor cells (NPCs) into specific types of neurons. In NPCs cultured primarily from the rat embryonic hippocampus, aucubin significantly elevated the number of GAD65/67 immunoreactive cells and the expression of GAD65/67 proteins was upregulated dramatically by more than three-fold at relatively low concentrations of aucubin (0.01 µM to 10 µM). The expression of both NeuN and vGluT1 of NPCs, the markers for neurons and glutamatergic cells, respectively, and the number of vGluT1 immunoreactive cells also increased with higher concentrations of aucubin (1 µM and 10 µM), but the ratio of the increases was largely lower than GAD expression and GAD immunoreactive cells. The GABAergic differentiation of pax6-expressing late NPCs into GABA-producing cells was further supported in cortical NPCs primarily cultured from transgenic mouse brains, which express recombinant GFP under the control of pax6 promoter. The results suggest that aucubin can be developed as a therapeutic candidate for neurodegenerative disorders caused by the loss of inhibitory GABAergic neurons.

Effect of aucubin on neural precursor cell survival during neuronal differentiation.

PURPOSE OF THE STUDY: Aucubin (ACB) is an iridoid glycoside with various biological activities. Previously, it is reported that ACB reduces cell survival and proliferation in many human tumors, whereas it facilitates cell survival and neuroprotection in damaged neuronal cells and disease models. However, its effects on cell survival in the non-proliferating or differentiated neurons are not known. MATERIALS AND METHODS: We examined whether ACB facilitated cell survival in differentiating neural precursor cells, HiB5, compared with the proliferating HiB5 cells at various concentrations. RESULTS: The cell viabilities were evaluated by measuring MTT values, cell numbers, amounts of neurotransmittersD1 and protein amounts of neuronal markers. Here, we showed that ACB promotes cell survival in differentiated neurons (10-200 µg/mL), but reduces it in proliferating NPCs (200-400 µg/mL). Protein amounts of neurofilament proteins, NF-H, NF-M, PSD-95 in post-synaptic density, GAP-43 in growing neurites and NeuN in differentiated neurons were upregulated by addition of ACB, indicating that cell survival increased in differentiated neurons, shown by immunoblot analysis. Especially, when PDGF was added into N2 media to facilitate neuronal differentiation of HiB5 cells, the viability of differentiated HiB5 cells was significantly elevated following the increase of ACB concentration. Furthermore, ACB promoted cell survival of specific neuron types, such as GABAergic neurons and glutamatergic neurons. When differentiated neurons were immunostained with markers for specific neurons, neuronal subtypes producing GABA and GAD 65/67 were immunostained more than subtypes producing glutamate and vGluT1. CONCLUSION: These results indicate that ACB improves neuronal cell survival in differentiated cells, suggesting it may be a therapeutic compound for neurodegenerative disorders.

Human Dental Pulp Stem Cells Are More Effective Than Human Bone Marrow-Derived Mesenchymal Stem Cells in Cerebral Ischemic Injury.

We compared the therapeutic effects and mechanism of transplanted human dental pulp stem cells (hDPSCs) and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in a rat stroke model and an in vitro model of ischemia. Rats were intravenously injected with hDPSCs or hBM-MSCs 24 h after middle cerebral artery occlusion (MCAo), and both groups showed improved functional recovery and reduced infarct volume versus control rats, but the hDPSC group showed greater reduction in infarct volume than the hBM-MSC group. The positive area for the endothelial cell marker was greater in the lesion boundary areas in the hDPSC group than in the hBM-MSC group. Administration of hDPSCs to rats with stroke significantly decreased reactive gliosis, as evidenced by the attenuation of MCAo-induced GFAP+/nestin+ and GFAP+/Musashi-1+ cells, compared with hBM-MSCs. In vivo findings were confirmed by in vitro data illustrating that hDPSCs showed superior neuroprotective, migratory, and in vitro angiogenic effects in oxygen-glucose deprivation (OGD)-injured human astrocytes (hAs) versus hBM-MSCs. Comprehensive comparative bioinformatics analyses from hDPSC- and hBM-MSC-treated in vitro OGD-injured hAs were examined by RNA sequencing technology. In gene ontology and KEGG pathway analyses, significant pathways in the hDPSC-treated group were the MAPK and TGF-ß signaling pathways. Thus, hDPSCs may be a better cell therapy source for ischemic stroke than hBM-MSCs.

Protein Interacting with Never in Mitosis A-1 Induces Glutamatergic and GABAergic Neuronal Differentiation in Human Dental Pulp Stem Cells.

INTRODUCTION: The purpose of this study was to investigate the role of protein interacting with never in mitosis A-1 (PIN1) in the neuronal or glial differentiation of human dental pulp stem cells (hDPSCs) and whether PIN1 can regulate determination of neuronal sub-phenotype. METHODS: After magnetic-activated cell sorting to separate CD34(+)/c-kit(+)/STRO-1(+) hDPSCs, cells were cultured in neurogenic medium. Differentiation was measured as Nissl staining and marker protein or mRNA expression by reverse transcriptase polymerase chain reaction, immunofluorescence, and flow cytometric analysis. RESULTS: PIN1 mRNA levels were upregulated in a time-dependent fashion during neurogenic differentiation. The PIN1 inhibitor juglone suppressed neuronal differentiation but promoted glial differentiation as assessed by the number of Nissl-positive cells and mRNA expression of neuronal markers (nestin, ßIII-tubulin, and NeuN) and a glial marker (glial fibrillary acidic protein). Conversely, overexpression of PIN1 by infection with adenovirus-PIN1 increased neuronal differentiation but decreased glial differentiation. Moreover, PIN1 overexpression increased the percentage of glutamatergic and GABAergic cells but decreased that of dopaminergic cells among total NeuN-positive hDPSCs. CONCLUSIONS: This is the first study to demonstrate that PIN1 overexpression induced glutamatergic and GABAergic neuronal differentiation but suppressed glial differentiation of hDPSCs, suggesting that enhancing PIN expression is important to obtain human glutamatergic and GABAergic neurons from hDPSCs.

Comparison of the effects of human dental pulp stem cells and human bone marrow-derived mesenchymal stem cells on ischemic human astrocytes in vitro.

This study assesses the cytoprotective effects of human dental pulp stem cells (hDPSCs) and conditioned medium from hDPSCs (CM-hDPSCs) on ischemic human astrocytes (hAs) in vitro compared with human bone marrow-derived mesenchymal stem cells (hMSCs). Ischemia of hAs was induced by oxygen-glucose deprivation (OGD). CM-hDPSCs and hMSCs were collected after 48 hr of culture. Cell death was determined by 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide and cellular ATP assays. The expression of glial fibrillary acidic protein (GFAP) and musashi-1 as markers of reactive astrogliosis was examined with immunochemical staining. mRNA expression and reactive oxygen species (ROS) were analyzed by RT-PCR and flow cytometry, respectively. OGD increased cytotoxicity in a time-dependent manner and decreased cellular ATP content concomitantly in hAs. Pretreatment and posttreatment with hDPSCs were associated with greater recovery from OGD-induced cytotoxicity in hAs compared with hMSCs. Similarly, CM-hDPSCs had a greater effect on OGD-induced cytotoxicity in a dose-dependent manner. Pre- and posttreatment with CM-hDPSCs or CM-hMSCs attenuated OGD-induced GFAP, nestin, and musashi-1 expression in hAs. Furthermore, treatment of cells with CM-hDPSCs and hMSCs blocked OGD-induced ROS production and interleukin-1ß upregulation. This study demonstrates for the first time that hDPSCs and CM-hDPSCs confer superior cytoprotection against cell death in an in vitro OGD model compared with hMSCs as shown by cell viability assay. Reactive gliosis, ROS production, and inflammatory mediators might contribute to this protective effect. Therefore, hDPSCs could represent an alternative source of cell therapy for ischemic stroke.

Long-term effects of magnetically targeted ferumoxide-labeled human neural stem cells in focal cerebral ischemia.

The long-term effect of magnetically targeted neural stem cells in a rat focal cerebral ischemia model was investigated. In middle cerebral artery occlusion (MCAO) stroke model rats, ferumoxide-labeled human neural stem cells (hNSCs) were injected into the tail vein. MCAO rats were divided into three groups: ischemia only (IO), ischemia with NSC injection (IC), and ischemia with NSC injection and the use of magnet targeting (IM). Four weeks after MCAO and 3 weeks posttransplantation, a greater number of hNSCs were found in ischemic lesion sites in IM rat brain compared with IO and IC animals. In addition, differentiation of hNSCs into neurons or astrocytes and angiogenesis were markedly increased. In IM rats, infarct volume was considerably reduced, and function was significantly improved. The present study indicates that long-term use of magnetic fields may be a useful way to improve the efficacy of targeted migration of stem cells and functional deficits in stem cell-based therapy for ischemic brain injury.

Gradient index lens based combined two-photon microscopy and optical coherence tomography.

We report a miniaturized probe-based combined two-photon microscopy (TPM) and optical coherence tomography (OCT) system. This system is to study the colorectal cancer in mouse models by visualizing both cellular and structural information of the colon in 3D with TPM and OCT respectively. The probe consisted of gradient index (GRIN) lenses and a 90° reflecting prism at its distal end for side-viewing, and it was added onto an objective lens-based TPM and OCT system. The probe was 2.2 mm in diameter and 60 mm in length. TPM imaging was performed by raster scanning of the excitation focus at the imaging speed of 15.4 frames/s. OCT imaging was performed by combining the linear sample translation and probe rotation along its axis. This miniaturized probe based dual-modal system was characterized with tissue phantoms containing fluorescent microspheres, and applied to image mouse colonic tissues ex vivo as a demonstration. As OCT and TPM provided structural and cellular information of the tissues respectively, this probe based multi-modal imaging system can be helpful for in vivo studies of preclinical animal models such as mouse colonic tumorigenesis.

15-hydroxyprostaglandin dehydrogenase in colorectal mucosa as a potential biomarker for predicting colorectal neoplasms.

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is downregulated during the early stages of colorectal carcinogenesis. The aim of the present study was to investigate the potential role of 15-PGDH in normal-appearing colorectal mucosa as a biomarker for predicting colorectal neoplasms. We obtained paired tumor and normal tissues from the surgical specimens of 32 sporadic colorectal cancer patients. mRNA expression of 15-PGDH was measured using a quantitative real-time PCR assay. We evaluated the association between 15-PGDH mRNA expression in normal-appearing mucosa, the presence of synchronous adenoma, and the cumulative incidence of metachronous adenoma. The relative 15-PGDH expression of normal-appearing mucosa in patients with synchronous adenoma was significantly lower than in patients without synchronous adenoma (0.71 vs 1.00, P = 0.044). The patients in the lowest tertile of 15-PGDH expression in normal-appearing mucosa were most likely to have synchronous adenoma (OR: 10.5, P = 0.024). Patients with low 15-PGDH expression in normal-appearing mucosa also demonstrated more advanced stage colorectal cancer (P = 0.045). However, there was no significant difference in the cumulative incidence of metachronous adenoma according to 15-PGDH mRNA expression in normal-appearing mucosa (P = 0.333). Hence, 15-PGDH in normal-appearing colorectal mucosa can be a useful biomarker of field effect for the prediction of sporadic synchronous neoplasms.

Inhibition of 15-hydroxyprostaglandin dehydrogenase by Helicobacter pylori in human gastric carcinogenesis.

Helicobacter pylori (H. pylori) infection induces a chronic inflammatory response, which promotes gastric carcinogenesis. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) plays a key role as a tumor suppressor in gastrointestinal cancers. The aim of this study was to elucidate the role of 15-PGDH in gastric carcinogenesis associated with H. pylori. 15-PGDH expression in gastric biopsies from H. pylori-infected (n = 25) and noninfected (n = 15) subjects was analyzed by quantitative real-time PCR, Western blot analysis, and immunohistochemistry. 15-PGDH DNA methylation was evaluated by methylation-specific PCR and pyrosequencing. The expression of 15-PGDH, Snail, extracellular signal-regulated kinase (ERK)1/2, TLR4, and MyD88 in response to H. pylori infection was assessed by immunoblot analysis. Compared with negative specimens, H. pylori-positive specimens had 2-fold lower 15-PGDH mRNA levels and significantly less 15-PGDH protein. In four H. pylori-infected subjects with longitudinal follow-up, the suppression of 15-PGDH expression was reversed by H. pylori eradication therapy. In parallel with suppressing 15-PGDH expression, H. pylori infection activated expression of TLR4 and MyD88 expression, increased levels of phospho-ERK1/2, and increased expression of EGF receptor (EGFR)-Snail. Inhibition of Snail and MyD88 reversed suppression of 15-PGDH expression, and siMyD88 reduced phosphorylated ERK1/2. Similarly, treatment with an ERK1/2 and EGFR inhibitor also restored 15-PGDH expression. H. pylori appeared to promote gastric carcinogenesis by suppressing 15-PGDH. This process is mediated by the TLR4/MyD88 pathway via ERK1/2 or EGFR-Snail transcriptional regulation. 15-PGDH may be a useful marker and a potential therapeutic target in H. pylori-induced gastric carcinogenesis.

Effects of duplicate administration of human neural stem cell after focal cerebral ischemia in the rat.

We investigated the functional and histological recovery of middle cerebral artery occluded (MCAo) rats after of duplicate intravenous (i.v.) injection of human neural stem cells (hNSCs). Rats received i.v. injections of hNSCs (HB1.F3, 4 × 10(6) cells) on day 1 (1C), day 7 (7C), or both days 1 and 7 (1/7C) following MCAo. Functional recovery of rats was evaluated 1 day before MCAo and 1, 7, 14, 21, and 28 days following MCAo, using the modified neurological severity score (mNSS), and cylinder test. Nissl staining and anti-human nuclear matrix antigen /NeuN or GFAP were used to measure infarct size and investigate the migration and differentiation of injected cells. Treatment with hNSCs did not significantly affect infarct size of ischemic animals. Behavior evaluation using mNSS showed that functional deficits in the 1C group were reduced faster than in the 7C and 1/7C groups, and functional recovery in 1/7C rats was significantly more pronounced than that in the 7C group (day 21). Injected cells were identified at the boundary of lesions, where they had differentiated into neurons and astrocytes. Our study suggests that duplicate i.v. administration of hNSCs after stroke offers no advantages over single administration, 1 day following an ischemic event.

Using a neodymium magnet to target delivery of ferumoxide-labeled human neural stem cells in a rat model of focal cerebral ischemia.

Efficiency of targeted delivery of stem cells via transplantation by intravenous injection is limited because of rapid clearance. Thus, more effective, newer methods are required. We hypothesized that combining the use of ferumoxide labeling and magnetic fields could enhance targeted delivery of stem cells. The effects of a magnetic field on proliferation, viability, and differentiation of human neural stem cells (NSCs) were determined in culture, and the results indicated that the difference between control and cultures exposed to a magnetic field were insignificant. To assess migration in vitro, ferumoxide-labeled cells were seeded into a culture dish that had a neodymium magnet below its center, and the labeled NSCs were found to aggregate above the magnet. To investigate targeted delivery of NSCs in vivo, rats were separated into three groups: ischemia only (IO), ischemia with injection of ferumoxide-labeled cells (IC), and ischemia with injection of labeled cells and magnet exposure (ICM). Twenty-four hours after middle cerebral artery occlusion (MCAo), labeled human NSCs were injected into the tail vein. Seven days after MCAo, ICM rats had a larger number and greater distribution of Prussian blue-positive NSCs as compared with controls. In addition, infarct volume in ICM rats was significantly reduced. Our study suggests that this use of a magnetic field may be useful for improving the efficacy of targeted migration of stem cells in stem-based cell therapy in ischemic brain injury.

MRI tracking of intravenously transplanted human neural stem cells in rat focal ischemia model.

The present study investigated the ability of 1.5 T clinical magnetic resonance imaging (MRI) to detect ferumoxides- labeled human neural stem cells (NSCs) that had been intravenously (i.v.) injected into a rat model of focal cerebral ischemia. To detect transplanted cells, hNSCs were labeled with ferumoxide then followed by bromodeoxyuridine (BrdU) prior to transplantation. In the rat ischemia-human NSC group, human NSCs (4 x 10(6)cells in 5 ml PBS) were injected via tail vein 24 h after middle cerebral artery occlusion (MCAo), and the brains of the rats were scanned using a 1.5 T MRI unit over a period of 4 weeks (1 day before MCAo, then 1 and 3 days after cell injection, and weekly thereafter). In histologic sections, transplanted cells were identified by Prussian blue and anti-BrdU fluorescence staining. Regions with hypointense signals on T2-weighted and 3D gradient echo MR images corresponded with areas stained by Prussian blue, which suggested the presence of superparamagnetic iron oxide (SPIO) nanoparticles within the engrafted cells. Hypointense areas on MR images were observed in peri-infarct areas 3 days after cell injection. The findings indicate that 1.5 T MRI has sufficient sensitivity to track engrafted stem cells in vivo.

Labeling efficacy of superparamagnetic iron oxide nanoparticles to human neural stem cells: comparison of ferumoxides, monocrystalline iron oxide, cross-linked iron oxide (CLIO)-NH2 and tat-CLIO.

OBJECTIVE: We wanted to compare the human neural stem cell (hNSC) labeling efficacy of different superparamagnetic iron oxide nanoparticles (SPIONs), namely, ferumoxides, monocrystalline iron oxide (MION), cross-linked iron oxide (CLIO)-NH(2) and tat-CLIO. MATERIALS AND METHODS: The hNSCs (5 x 10(5) HB1F3 cells/ml) were incubated for 24 hr in cell culture media that contained 25 microg/ml of ferumoxides, MION or CLIO-NH(2), and with or without poly-L-lysine (PLL) and tat-CLIO. The cellular iron uptake was analyzed qualitatively with using a light microscope and this was quantified via atomic absorption spectrophotometry. The visibility of the labeled cells was assessed with MR imaging. RESULTS: The incorporation of SPIONs into the hNSCs did not affect the cellular proliferations and viabilities. The hNSCs labeled with tat-CLIO showed the longest retention, up to 72 hr, and they contained 2.15+/-0.3 pg iron/cell, which are 59 fold, 430 fold and six fold more incorporated iron than that of the hNSCs labeled with ferumoxides, MION or CLIO-NH(2), respectively. However, when PLL was added, the incorporation of ferumoxides, MION or CLIO-NH(2) into the hNSCs was comparable to that of tat-CLIO. CONCLUSION: For MR imaging, hNSCs can be efficiently labeled with tat-CLIO alone or with a combination of ferumoxides, MION, CLIO-NH(2) and the transfection agent PLL.

Effect of 17-alpha-ethynylestradiol on germ cell proliferation in organ and primary culture of medaka (Oryzias latipes) testis.

Organ cultures and primary cell cultures of medaka (Oryzias latipes) testis were compared with respect to cell viability and cell proliferation. The analysis by fluorescence microscopy and flow cytometry showed that in both cultures, the cells remained viable for at least 1 day and cell proliferation could be analyzed reliably by BrdU incorporation. The proliferating cells were mostly spermatogonia located at the periphery of the testis in tissue sections. Both culture systems were used to study the effect of 17-alpha-ethynylestradiol on cell proliferation. The results obtained with organ and primary cultures were consistent: low concentrations (0.01 and 1 nm) of synthetic estrogen stimulated cell proliferation slightly, while a higher concentration (100 nm) had an inhibitory effect. Both culture methods are suitable for the analysis of substances that might interfere with germ cell proliferation or other functions in spermatogenesis.

Primary culture of medaka (Oryzias latipes) testis: a test system for the analysis of cell proliferation and differentiation.

An in vitro test system using primary testis cells of the medaka (Oryzias latipes) was established that provides quantitative data on cell proliferation and spermatocyte differentiation. The primary cultures were characterised over a period of 2 days with respect to cell viability and the distribution of adherent and suspended cells. These two cell populations were maintained at a dynamic equilibrium in vitro for several days. The proliferating cells were predominantly present amongst the clusters of suspended cells as determined by BrdU labelling (cytological identification and quantification by ELISA). Based on cytological criteria the proliferating cells were mostly spermatogonia and preleptotene spermatocytes. Differentiation of spermatocytes to spermatids or spermatozoa was also observed mainly amongst suspended cells. Quantification of cell proliferation and cell differentiation by flow cytometry was achieved by labelling the primary cells with carboxyfluorescein diacetate N-succinimidyl ester, which allowed the identification and quantification of meiotically or mitotically dividing primary cells. Addition of the flavonoid genistein (10 microg/ml) to the primary cultures inhibited both cell proliferation and cell differentiation significantly. The test system is suitable for the study of the effect of substances which interfere with spermatogenesis in the vertebrate medaka model.