Non-labeled Cells Research Articles

Differential gene expression and transport functionality in the bundle sheath versus mesophyll - a potential role in leaf mineral homeostasis.

Under fluctuating ambient conditions, the ability of plants to maintain hydromineral homeostasis requires the tight control of long distance transport. This includes the control of radial transport within leaves, from veins to mesophyll. The bundle sheath is a structure that tightly wraps around leaf vasculature. It has been suggested to act as a selective barrier in the context of radial transport. This suggestion is based on recent physiological transport assays of bundle sheath cells (BSCs), as well as the anatomy of these cells.We hypothesized that the unique transport functionality of BSCs is apparent in their transcriptome. To test this, we compared the transcriptomes of individually hand-picked protoplasts of GFP-labeled BSCs and non-labeled mesophyll cells (MCs) from the leaves of Arabidopsis thaliana. Of the 90 genes differentially expressed between BSCs and MCs, 45% are membrane related and 20% transport related, a prominent example being the proton pump AHA2. Electrophysiological assays showed that the major AKT2-like membrane K+ conductances of BSCs and MCs had different voltage dependency ranges. Taken together, these differences may cause simultaneous but oppositely directed transmembrane K+ fluxes in BSCs and MCs, in otherwise similar conditions.

Cultured eastern oysters (Crassostrea virginica): retention and assimilation of picophytoplankton using a multi-biomarker approach

In this study, we investigated the food sources of eastern oysters Crassostrea virginica cultivated in Atlantic Canada. Stable isotopes (13 C and 15 N) and fatty acid biomarkers were used to identify these sources under in situ conditions for suspended (∼0.5 m below surface) and bottom (∼2 m) culture stocks. It was found that particulate organic matter represented the main food source, with major contributions from live phytoplankton. Higher lipid contents were detected in the digestive glands of suspended oysters compared to bottom oysters (p , mean ± SEM) compared to nanophytoplankton biomass (NPP, >2 μm, 1.05 ± 0.15 μg l−1 , mean ± SEM). To determine whether the small size PPP was captured and assimilated by C. virginica , feeding trials were conducted in the laboratory using three PPP/NPP diets (20%, 50%, and 80% PPP), consisting of isotopically-labelled (δ13 C) PPP cells (Nannochloropsis oculata ) and non-labelled NPP cells (Tisochrysis lutea ). An isotopically-labelled fatty acids analysis indicated PPP assimilation in various tissues (digestive gland, gills, mantle, and abductor muscle), including from oysters fed the reduced (20%) PPP diet. Isotopic enrichment (13 C) in the FA 22:2 (non-methylene-interrupted or NMI) showed that precursors of NMIs utilized PPP carbon in its biosynthesis process. In conclusion, C. virginica assimilated primarily particulate organic matter (POM), including PPP, which dominated the phytoplankton community in near surface waters. C. virginica can exploit PPP carbon during fatty acid production and further biosynthesis.

Random Allocation of Blastomere Descendants to the Trophectoderm and ICM of the Bovine Blastocyst.

The first lineage specification during mammalian embryo development can be visually distinguished at the blastocyst stage. Two cell lineages are observed on the embryonic-abembryonic axis of the blastocyst: the inner cell mass and the trophectoderm. The timing and mechanisms driving this process are still not fully understood. In mouse embryos, cells seem prepatterned to become certain cell lineage because the first cleavage plane has been related with further embryonic-abembryonic axis at the blastocyst stage. Nevertheless, this possibility has been very debatable. Our objective was to determine whether this would be the case in another mammalian species, the bovine. To achieve this, cells of in vitro produced bovine embryos were traced from the 2-cell stage to the blastocyst stage. Blastocysts were then classified according to the allocation of the labeled cells in the embryonic and/or abembryonic part of the blastocyst. Surprisingly, we found that there is a significant percentage of the embryos (∼60%) with labeled and nonlabeled cells randomly distributed and intermingled. Using time-lapse microscopy, we have identified the emergence of this random pattern at the third to fourth cell cycle, when cells started to intermingle. Even though no differences were found on morphokinetics among different embryos, these random blastocysts and those with labeled cells separated by the embryonic-abembryonic axis (deviant pattern) are significantly bigger; moreover deviant embryos have a significantly higher number of cells. Interestingly, we observed that daughter cells allocation at the blastocyst stage is not affected by biopsies performed at an earlier stage.

Parametric analysis of colony morphology of non-labelled live human pluripotent stem cells for cell quality control.

Given the difficulties inherent in maintaining human pluripotent stem cells (hPSCs) in a healthy state, hPSCs should be routinely characterized using several established standard criteria during expansion for research or therapeutic purposes. hPSC colony morphology is typically considered an important criterion, but it is not evaluated quantitatively. Thus, we designed an unbiased method to evaluate hPSC colony morphology. This method involves a combination of automated non-labelled live-cell imaging and the implementation of morphological colony analysis algorithms with multiple parameters. To validate the utility of the quantitative evaluation method, a parent cell line exhibiting typical embryonic stem cell (ESC)-like morphology and an aberrant hPSC subclone demonstrating unusual colony morphology were used as models. According to statistical colony classification based on morphological parameters, colonies containing readily discernible areas of differentiation constituted a major classification cluster and were distinguishable from typical ESC-like colonies; similar results were obtained via classification based on global gene expression profiles. Thus, the morphological features of hPSC colonies are closely associated with cellular characteristics. Our quantitative evaluation method provides a biological definition of ‘hPSC colony morphology’, permits the non-invasive monitoring of hPSC conditions and is particularly useful for detecting variations in hPSC heterogeneity.

Morphological Evaluation of Nonlabeled Cells to Detect Stimulation of Nerve Growth Factor Expression by Lyconadin B

The success of drug development is greatly influenced by the efficiency of drug screening methods. Recently, phenotype-based screens have raised expectations, based on their proven record of identifying first-in-class drugs at a higher rate. Although fluorescence images are the data most commonly used in phenotype-based cell-based assays, nonstained cellular images have the potential to provide new descriptive information about cellular responses. In this study, we applied morphology-based evaluation of nonlabeled microscopic images to a phenotype-based assay. As a study case, we attempted to increase the efficiency of a cell-based assay for chemical compounds that induce production of nerve growth factor (NGF), using lyconadin B as a model compound. Because the total synthesis of lyconadin B was accomplished very recently, there is no well-established cell-based assay scheme for further drug screening. The conventional cell-based assay for evaluating NGF induction requires two types of cells and a total of 5 days of cell culture. The complexity and length of this assay increase both the risk of screening errors and the cost of screening. Our findings show that analysis of cellular morphology enables evaluation of NGF induction by lyconadin B within only 9 h.

The ontogeny of choanocyte chambers during metamorphosis in the demosponge Amphimedon queenslandica.

BackgroundThe aquiferous body plan of poriferans revolves around internal chambers comprised of choanocytes, a cell type structurally similar to choanoflagellates. These choanocyte chambers perform a range of physiological and developmental functions, including the capture of food and the generation of stem cells. Despite the increasing interest for choanocytes as sponge stem cells, there is limited knowledge on the development of choanocyte chambers. Using a combination of cell lineage tracing, antibody staining and EdU labeling, here we examine the development of choanocytes and the chambers they comprise during metamorphosis in the marine demosponge Amphimedon queenslandica.ResultsLineage-tracing experiments show that larval epithelial cells transform into mesenchymal pluripotent stem cells, resembling archeocytes, within 24 h of initiating metamorphosis. By 36 h, some of these labeled archeocyte-like cells have differentiated into choanocytes that will form the first postlarval choanocyte chambers. Non-labeled cells also contribute to these primary choanocyte chambers, consistent with these chambers being a chimera of multiple transdifferentiated larval cell types and not the proliferation of a single choanocyte precursor. Moreover, cell proliferation assays demonstrate that, following the initial formation of choanocyte chambers, chambers grow at least partially by the proliferation of choanocytes within the chamber, although recruitment of individual cells into established chambers also appears to occur. EdU labeling of postlarvae and juveniles reveals that choanocyte chambers are the primary location of cell proliferation during metamorphosis.ConclusionOur results show that multiple larval cell lineages typically contribute to formation of individual choanocyte chambers at metamorphosis, contrary to previous reports in other species that show sponge choanocyte chambers form clonally. Choanocytes in postlarval and juvenile A. queenslandica chambers can also divide, with choanocyte chambers being the primary location of cell proliferation. Interestingly, the level of cell proliferation varies greatly between chambers and appears to be contingent on the size, location and developmental state of the chamber. Small chambers on the periphery of the body tend to possess more dividing cells. As choanocytes can also dedifferentiate into archeocyte-like cells, cell proliferation in chambers may not only contribute to chamber growth and self-renewal but also increase the number of pluripotent archeocytes.

Abstract B69: VAL401 decreases tumor volume in a xenograft model of pancreatic cancer utilizing a novel improved luciferase labelling technique

Abstract Bioluminescent-labelling allows sensitive non-invasive sequential imaging of tumor development and early metastasis. Current methods for the genetic modification of cells typically use integrating genotoxic viruses that can potentially disrupt the molecular behavior of cancer cell lines due to their random nature of integration. VAL401 is the reformulation of a clinical drug to enable use in the treatment of cancer. Preclinical data indicate potential use of the reformulated drug in lung cancer, where many subsets of patients have currently a high unmet medical need. We have utilized a non-viral DNA vector that comprises an S/MAR (Scaffold/Matrix Attachment Region) element to stably modify cells to be further used in xenograft studies to allow long term expression without affecting cell behavior or silencing over cell divisions. Human BxPC3 pancreatic cancer cells were stably transfected with a pSMARt-UBC-Luc and cultured for 4 weeks under selection. Colonies that formed after this period were isolated and expanded in normal medium and evaluated for luciferase expression and molecular integrity of the DNA vector. For in vivo studies, BxPC3 cells were inoculated orthotopically into the pancreas of athymic nude mice. Four experimental groups were included in the study: 1) Control group receiving vehicle; 2) Reference compound gemcitabine (60 mg/kg, q3dx4 i.p, one week pause, q3dx4 i.p.); 3) Test compound VAL401 (1 mg/kg, p.o. daily); 4) Test compound VAL401 (2 mg/kg, p.o. daily). The luciferase transfected cells maintained their original properties with stable expression. Luciferase-labelled BxPC3 cells inoculated orthotopically into the pancreas were successfully followed for 5 weeks with non-invasive bioluminescence imaging by IVIS, and the results demonstrated high-quality follow-up of tumor growth compared with tumor models using non-labelled cells. BxPC3-luc cells induced growth of pancreatic tumors with high take rate in all groups. Gemcitabine and both studied doses of VAL401 decreased tumor volume, and the same trend was seen in tumor weight and the BLI parameters (total flux, area and average radiance) during the study. In conclusion, both gemcitabine and VAL401 decreased tumor volume and same trend was seen in BLI. Our results demonstrated that S/MAR DNA vectors are able to produce genetically modified cells without the limitations of random genomic integration, whilst providing extra-chromosomal mitotic stability and sustained transgene expression at high level. When utilized in orthotopic xenograft studies, these luciferase expressing cells formed a reliable and essential non-invasive imaging platform that improves substantially efficacy testing of anticancer drug candidates. Citation Format: Mari I. Suominen, Jenni Bernoulli, Suzanne Dilly, Johanna Tuomela, Matthias Bozza, Katja M. Fagerlund, George Morris, Jussi M. Halleen, Richard Harbottle. VAL401 decreases tumor volume in a xenograft model of pancreatic cancer utilizing a novel improved luciferase labelling technique. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B69.

Intravenous Bone Marrow Stem Cell Grafts Preferentially Migrate to Spleen and Abrogate Chronic Inflammation in Stroke.

Background and Purpose—Adult stem cell therapy is an experimental stroke treatment. Here, we assessed homing and anti-inflammatory effects of bone marrow stromal cells (hBMSCs) in chronic stroke.Methods—At 60 days post stroke, adult Sprague–Dawley rats received intravenous hBMSCs (4×106 labeled or nonlabeled cells) or vehicle (saline). A sham surgery group served as additional control. In vivo imaging was conducted between 1 hour and 11 days post transplantation, followed by histological examination.Results—Labeled hBMSCs migrated to spleen which emitted significantly higher fluorescent signal across all time points, especially during the first hour, and were modestly detected in the head region at the 12 hours and 11 days, compared with nonlabeled hBMSCs and vehicle-infused stroke animals, or sham (P<0.05). At 11 days post transplantation, ex vivo imaging confirmed preferential hBMSC migration to the spleen over the brain. Hematoxylin and eosin staining revealed significant 15% and 30% reductions in striatal infarct and peri-infarct area, and a trend of rescue against neuronal loss in the hippocampus. Unbiased stereology showed significant 75% and 60% decrements in major histocompatibility complex II–activated inflammatory cells in gray and white matter, and a 43% diminution in tumor necrosis factor-α cell density in the spleen of transplanted stroke animals compared with vehicle-infused stroke animals (P<0.05). Human antigen immunostaining revealed 0.03% hBMSCs survived in spleen and only 0.0007% in brain. MSC migration to spleen, but not brain, inversely correlated with reduced infarct, peri-infarct, and inflammation.Conclusions—hBMSC transplantation is therapeutic in chronic stroke possibly by abrogating the inflammation-plagued secondary cell death.

Abstract 3246: Novel luciferase labelling technique to improve imaging of orthotopic prostate and pancreatic cancer models

Abstract Bioluminescent-labelling allows sensitive non-invasive sequential imaging of tumor development and early metastasis. However, current methods for the genetic modification of cells typically use integrating genotoxic viruses that can potentially disrupt the molecular behavior of cancer cell lines due to their random nature of integration. Here, we utilized a non-viral DNA vector that comprises an S/MAR (Scaffold/Matrix Attachment Region) element to stably modify cells to be further used in xenograft studies to allow long term expression without affecting cell behavior or silencing over cell divisions. Human PC-3 prostate cancer cells and BxPC-3 pancreatic cancer cells were stably transfected with a pCAG-LUC-S/MAR and cultured for 4 weeks under selection. Colonies that formed after this period were isolated and expanded in normal medium and evaluated for luciferase expression and molecular integrity of the DNA vector. For in vivo studies, PC-3 cells were inoculated orthotopically into the prostate, and BxPC-3 cells into the pancreas using athymic and BALB/c nude mice. For comparison, similar experiments with the corresponding study designs were performed with the non-labelled parental cell lines. The luciferase transfected cells maintained their original properties with stable expression. Luciferase-labelled PC-3 and BxPC-3 cells inoculated orthotopically into the prostate and pancreas, respectively, were successfully followed for 5 weeks with non-invasive bioluminescence imaging by IVIS. The results demonstrated high-quality follow-up of tumor growth compared with tumor models using non-labelled cells. In conclusion, S/MAR DNA vectors are able to produce genetically modified cells without the limitations of random genomic integration, whilst providing extra-chromosomal mitotic stability and sustained transgene expression at high level. When utilized in orthotopic xenograft studies, these luciferase expressing cells formed a reliable and essential non-invasive imaging platform that improves substantially efficacy testing of anticancer drug candidates. Citation Format: Jenni Bernoulli, Johanna Tuomela, Matthias Bozza, Katja M. Fagerlund, Mari I. Suominen, George Morris, Jussi M. Halleen, Richard Harbottle. Novel luciferase labelling technique to improve imaging of orthotopic prostate and pancreatic cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3246. doi:10.1158/1538-7445.AM2015-3246

Development of a Monitoring Method for Nonlabeled Human Pluripotent Stem Cell Growth by Time-Lapse Image Analysis.

: Cell growth is an important criterion for determining healthy cell conditions. When somatic cells or cancer cells are dissociated into single cells for passaging, the cell numbers can be counted at each passage, providing information on cell growth as an indicator of the health conditions of these cells. In the case of human pluripotent stem cells (hPSCs), because the cells are usually dissociated into cell clumps of ∼50-100 cells for passaging, cell counting is time-consuming. In the present study, using a time-lapse imaging system, we developed a method to determine the growth of hPSCs from nonlabeled live cell phase-contrast images without damaging these cells. Next, the hPSC colony areas and number of nuclei were determined and used to derive equations to calculate the cell number in hPSC colonies, which were assessed on time-lapse images acquired using a culture observation system. The relationships between the colony areas and nuclei numbers were linear, although the equation coefficients were dependent on the cell line used, colony size, colony morphology, and culture conditions. When the culture conditions became improper, the change in cell growth conditions could be detected by analysis of the phase-contrast images. This method provided real-time information on colony growth and cell growth rates without using treatments that can damage cells and could be useful for basic research on hPSCs and cell processing for hPSC-based therapy. This is the first study to use a noninvasive method using images to systemically determine the growth of human pluripotent stem cells (hPSCs) without damaging or wasting cells. This method would be useful for quality control during cell culture of clinical hPSCs.

Salivary gland homeostasis is maintained through acinar cell self-duplication.

Current dogma suggests that salivary gland homeostasis is stem cell dependent. However, the extent of stem cell contribution to salivary gland maintenance has not been determined. We investigated acinar cell replacement during homeostasis, growth, and regeneration, using an inducible CreER(T2) expressed under the control of the Mist1 gene locus. Genetic labeling, followed by a chase period, showed that acinar cell replacement is not driven by the differentiation of unlabeled stem cells. Analysis using R26(Brainbow2.1) reporter revealed continued proliferation and clonal expansion of terminally differentiated acinar cells in all major salivary glands. Induced injury also demonstrated the regenerative potential of pre-labeled acinar cells. Our results support a revised model for salivary gland homeostasis based predominantly on self-duplication of acinar cells, rather than on differentiation of stem cells. The proliferative capacity of differentiated acinar cells may prove critical in the implementation of cell-based strategies to restore the salivary glands.

α-Transducin and α-gustducin immunoreactive cells in the stomach of common sole (Solea solea) fed with mussel meal.

Vertebrates perceive a variety of exogenous substances using two main chemosensory systems, taste and olfaction. The taste perception occurs through the interaction of taste receptors associated with specific G protein subunits such as α-transducin (Gαtran) and α-gustducin (Gαgust). Aquatic vertebrates are also provided with a chemosensory system consisting of solitary chemosensory cells distributed to the oropharynx and skin. In this study, we identified Gαtran and Gαgust-immunoreactive cells intermingled with non-labeled epithelial cells in the gastric mucosa of the common sole. A long-term diet with increasing concentrations of mussel meal in the protein component of a conventional fish meal-based diet induced a dose-dependent increase in the gastric epithelial area and density of Gαtran and Gαgust immunoreactive cells. These findings suggest that taste-related molecules are regulated by changes in diet formulation in common sole aquaculture.

Effect of tamoxifen and raloxifene on the proliferative activity of the breast epithelium in premenopausal women.

OBJECTIVESTo compare the effects of tamoxifen and raloxifene on the proliferative activity of normal breast tissue in premenopausal women as measured by Ki-67/MIB-1 expression.STUDY DESIGNA total of 48 women with benign breast nodules and a recommendation for surgical removal of the lesion took part in this study. They were randomized to use tamoxifen or raloxifene for 22 days, after which they were submitted to surgery. During the surgical procedure, a 1-cm fragment of normal breast tissue was removed to study Ki-67 expression.RESULTSThe mean percentage ratios between immunolabeled and non-labeled cells were 2.02 ± 1.09 and 3.13 ± 3.23 for the tamoxifen and raloxifene groups, respectively. There was no statistically significant difference between the tamoxifen (n = 16) and raloxifene (n = 14) groups in relation to the immunohistochemical analysis of Ki-67 (P = 0.205).CONCLUSIONThe results of this study showed no difference between tamoxifen and raloxifene with respect to the potential of these drugs to reduce the proliferative activity of the normal breast epithelium in premenopausal women.

78 NONINVASIVE CELL LINEAGE TRACING IN BOVINE EMBRYOS FROM 2-CELL STAGE UP TO BLASTOCYST STAGE

The first lineage specification occurs during pre-implantation mammalian development. At the blastocyst stage, 2 cell lineages can be distinguished: the inner cell mass (ICM) and the trophectoderm (TE). The exact timing when embryo cells are skewed to these lineages is not clearly determined in mammalian species. In murine embryos, it has been suggested that the first cleavage plane might be related to the embryonic-abembryonic (Em-Ab) axis at blastocyst stage. Thus, the daughter cells of the 2-cell embryo might already be predisposed to a specific cell lineage further on development. The objective of the present study was to observe how the first cleavage in bovine embryos may be related to cell lineage allocation at the blastocyst stage, using a noninvasive tracing approach. Bovine oocytes were harvested, in vitro matured, and fertilised. At the 2-cell stage, embryos were injected in one blastomere with the membrane tracer DiI. At the blastocyst stage, embryos (n = 346) were classified as orthogonal when the Em-Ab axis was orthogonally divided by the borderline between labelled and non-labelled cells; as deviant if the borderline was overlapping the Em-Ab axis; and as random when the labelled and non-labelled cells were randomly distributed. Total cell count (TCC) and the ICM/TE ratio was allowed by DNA staining with 4′,6-diamidino-2-phenylindole (DAPI) and by immunostaining of the ICM with Sox2 antibody. Analysis of variance was performed by one-way ANOVA employing IBM SPSS v21 (SPSS Inc., Chicago, IL, USA) to determine any difference between the cell lineage allocation patterns, TCC, and the ICM/TE ratio. P-values = 0.05 were considered significant. All values are reported as mean ± standard error of mean. Within 40 repetitions, the blastocyst classification was as follows: orthogonal 14.9% (±2.32, n = 56), deviant 22.2% (±2.58, n = 80), and random 62.9% (±2.64, n = 210). A significant difference was found in the incidence between the random group against the orthogonal and deviant, but not between the latter two. Regarding TCC, a significant difference was observed only between the orthogonal (99.6 ± 11.7 cells, n = 15) and deviant (135 ± 7.3 cells, n = 25) groups, but not with random embryos (116 ± 5.5 cells, n = 42). Finally, no significant difference was found among the groups concerning the ICM/TE ratio (0.43 ± 0.07 for orthogonal, n = 7; 0.54 ± 0.06 for deviant, n = 14; and 0.40 ± 0.03 for random embryos, n = 26). In conclusion, bovine embryos present a marked tendency for a random distribution of the daughter cells derived from the 2-cell blastomeres. However, around 37% of the blastocysts present a patterned cell division, where the daughter cells remain together through pre-implantation development. The effect of these cell lineage allocation patterns on implantation and further embryo development needs to be addressed.The authors acknowledge Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73) and CONACyT Mexico for funding.

Digital cell counting device integrated with a single-cell array.

In this paper, we present a novel cell counting method accomplished using a single-cell array fabricated on an image sensor, complementary metal oxide semiconductor sensor. The single-cell array was constructed using a microcavity array, which can trap up to 7,500 single cells on microcavities periodically arranged on a plane metallic substrate via the application of a negative pressure. The proposed method for cell counting is based on shadow imaging, which uses a light diffraction pattern generated by the microcavity array and trapped cells. Under illumination, the cell-occupied microcavities are visualized as shadow patterns in an image recorded by the complementary metal oxide semiconductor sensor due to light attenuation. The cell count is determined by enumerating the uniform shadow patterns created from one-on-one relationships with single cells trapped on the microcavities in digital format. In the experiment, all cell counting processes including entrapment of non-labeled HeLa cells from suspensions on the array and image acquisition of a wide-field-of-view of 30 mm2 in 1/60 seconds were implemented in a single integrated device. As a result, the results from the digital cell counting had a linear relationship with those obtained from microscopic observation (r2 = 0.99). This platform could be used at extremely low cell concentrations, i.e., 25–15,000 cells/mL. Our proposed system provides a simple and rapid miniaturized cell counting device for routine laboratory use.

Abstract P4-11-03: Effect of tamoxifen and raloxifene on proliferative activity of breast epithelium in premenopausal women

Abstract Background: Both Tamoxifen and raloxifene have been demonstrated to be effective to reduce the incidence of breast cancer among women. Objective: To compare the effects of raloxifene (60 mg/day) and tamoxifen (20 mg/day) on the proliferative activity of normal human breast epithelium, during the luteal phase of the menstrual cycle, by means of the immunoexpression of Ki-67/MIB-1. Patients and Methods: Forty-eight women presenting benign breast nodules with an indication for lesion excision took part in this study. These women were randomized to use tamoxifen or raloxifene for 22 days. They underwent surgery after using the medication, and a one-centimeter fragment of normal breast tissue was removed to study the immunoexpression of Ki-67. For this, the frequency of cells immunolabeled for Ki-67/MIB-1 per thousand epithelial cells was quantified. Results: The mean percentage ratios between immunolabeled and non-labeled cells were 2.02 (±1.09) and 3.13 (±3.23) for the tamoxifen and raloxifene groups, respectively. There was no statistically significant difference between the two groups, tamoxifen (n = 16) and raloxifene (n = 14), in relation to the immunoexpression of Ki-67 (p = 0.205). The mean of progesterone level was 19.62 (±17.67) for the tamoxifen group and 14.59 (±8.98) for the raloxefene group (p = 0.32). The plasma levels of estradiol, the values found were: 355.72 (± 337.98) and 171.02 (± 119.83) for groups of tamoxifen e raloxifene groups, respectively (p = 0.055). Conclusion: In this study, we observed that there was no difference in the potential for reducing the proliferative activity of normal breast epithelium between tamoxifen and raloxifene in premenopausal women. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-11-03.

Labeling of islet cells with iron oxide nanoparticles through DNA hybridization for highly sensitive detection by MRI

A labeling method for islet cells with superparamagnetic iron oxide nanoparticles (SPIOs) based on DNA hybridization is proposed for monitoring of transplanted islets by magnetic resonance imaging (MRI). The surfaces of SPIOs were modified by via Michael reaction by reacting oligo-(deoxyadenylic acid)-bearing a terminal thiol group at the 5′-end ((dA)20-SH) with maleic acid functional groups on the SPIOs. The SPIOs were immobilized on islet cells which had been pretreated with oligo-(thymidylic acid)–poly(ethylene glycol)–phospholipid conjugates ((dT)20–PEG–DPPE) through DNA hybridization. Transmission electron microscopy observations revealed that SPIOs were initially anchored on the islet cell surfaces and subsequently transferred to endosomes or exfoliated with time. The SPIO-labeled islet cells could be clearly detected as dark spots by T2∗-weighted MR image, whereas non-labeled islet cells could not be detected.

Superparamagnetic Iron Oxide is Suitable to Label Tendon Stem Cells and Track Them In Vivo with MR Imaging

Tendon stem cells (TSCs) may be used to effectively repair or regenerate injured tendons. However, the fates of TSCs once implanted in vivo remain unclear. This study was aimed to determine the feasibility of labeling TSCs with super-paramagnetic iron oxide (SPIO) nano-particles to track TSCs in vivo using MRI. Rabbit TSCs were labeled by incubation with 50μg/mL SPIO. Labeling efficiency, cell viability, and proliferation were then measured, and the stemness of TSCs was tested by quantitative real time RT-PCR (qRT-PCR) and immunocytochemistry. We found that the labeling efficiency of TSCs reached as high as 98%, and that labeling at 50μg/mL SPIO concentrations did not alter cell viability and cell proliferation compared to non-labeled control cells. Moreover, the expression levels of stem cell markers (Nucleostemin, Nanog, and Oct-4) did not change in SPIO-labeled TSCs compared to non-labeled cells. Both labeled and non-labeled cells also exhibited similar differentiation potential. Finally, labeled TSCs could be detected by MRI both in vitro and in vivo. Taken together, the findings of this study show that labeling TSCs with SPIO particles is a feasible approach to track TSCs in vivo by MRI, which offers a non-invasive method to monitor repair of injured tendons.

Round-tip dielectrophoresis-based tweezers for single micro-object manipulation

In this paper, we present an efficient methodology to manipulate a single micro-object using round-tip positive dielectrophoresis-based tweezers. The tweezers consist of a glass needle with a round-tip and a pair of thin gold-film electrodes. The round-tip, which has a radius of 3µm, is formed by melting a finely pulled glass needle and concentrates the electric field at the tip of the tweezers, which allows the individual manipulation of single micro-objects. The tweezers successfully captured, conveyed, and positioned single cell-sized liposomes with diameters of 5–23µm, which are difficult to manipulate with conventional manipulation methodologies, such as optical tweezers or glass micropipettes, due to the similarities between their optical properties and those of the media, as well as the ease with which they are deformed or broken. We used Stokes’ drag theory to experimentally evaluate the positive dielectrophoresis (pDEP) force generated by the tweezers as a function of the liposome size, the content of the surrounding media, and the applied AC voltage and frequency. The results agreed with the theoretically deduced pDEP force. Finally, we demonstrated the separation of labeled single cells from non-labeled cells with the tweezers. This device can be used as an efficient tool for precisely and individually manipulating biological micro-objects that are typically transparent and flexible.

Effect of dietary treatment with dimethylarsinous acid (DMAIII) on the urinary bladder epithelium of arsenic (+3 oxidation state) methyltransferase (As3mt) knockout and C57BL/6 wild type female mice

Chronic exposure to inorganic arsenic (iAs) is carcinogenic to the human urinary bladder. It produces urothelial cytotoxicity and proliferation in rats and mice. DMAV, a major methylated urinary metabolite of iAs, is a rat bladder carcinogen, but without effects on the mouse urothelium. DMAIII was shown to be the likely urinary metabolite of DMAV inducing urothelial changes and is also postulated to be one of the active metabolites of iAs. To evaluate potential DMAIII-induced urothelial effects, it was administered to As3mt knockout mice which cannot methylate arsenicals. Female C57BL/6 wild type and As3mt knockout mice (10/group) were administered DMAIII, 77.3ppm in water for four weeks. Urothelial effects were evaluated by light and scanning electron microscopy (EM) and immunohistochemical detection of bromodeoxyuridine (BrdU) incorporation. EM findings were rated 1–5, with higher rating indicating greater extent of cytotoxicity visualized. DMAIII significantly increased the BrdU labeling index, a ratio of BrdU labeled cells to non-labeled cells, in the treated knockout group compared to control and wild type treated groups. DMAIII induced simple hyperplasia in more knockout mice (4/10) compared to wild type mice (2/10). All treated knockout mice had more and larger intracytoplasmic granules compared to the treated wild type mice. Changes in EM classification were not significant. In conclusion, DMAIII induces urothelial toxicity and regenerative hyperplasia in mice and most likely plays a role in inorganic arsenic-induced urothelial changes. However, DMAV does not induce hyperplasia in mice, suggesting that urinary concentrations of DMAIII do not reach cytotoxic levels in DMAV-treated mice.