In vitro study of CD133 human stem cells labeled with superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs) are applied in stem cell labeling because of their high magnetic susceptibility as compared with ordinary paramagnetic species, their low toxicity, and their ease of magnetic manipulation. The present work is the study of CD133(+) stem cell labeling by SPIONs coupled to a specific antibody (AC133), resulting in the antigenic labeling of the CD133+ stem cell, and a method was developed for the quantification of the SPION content per cell, necessary for molecular imaging optimization. Flow cytometry analysis established the efficiency of the selection process and helped determine that the CD133 cells selected by chromatographic affinity express the transmembrane glycoprotein CD133. The presence of antibodies coupled to the SPION, expressed in the cell membrane, was observed by transmission electron microscopy. Quantification of the SPION concentration in the marked cells using the ferromagnetic resonance technique resulted in a value of 1.70 x 10 (13) mol iron (9.5 pg) or 7.0 x 10 (6) nanoparticles per cell ( the measurement was carried out in a volume of 2 mu L containing about 6.16 x 10 5 pg iron, equivalent to 4.5 x 10 (11) SPIONs). (c) 2008 Elsevier Inc. All rights reserved.

Ultrastructural characterization of CD133(+) stem cells bound to superparamagnetic nanoparticles: possible biotechnological applications

CD133 antigen is an integral membrane glycoprotein that can bind with different cells. Originally, however. this cellular surface antigen was expressed in human stem cells and in various cellular progenitors of the haematopoietic system. Human cord blood has been described as an excellent source of CD133(+) haematopoietic progenitor cells with a large application potential. One of the main objectives of the present study is to describe for the first time the ultrastructural characteristics of CD133(+) stem cells using transmission electronic microscopy. Another objective of the manuscript is to demonstrate through transmission electronic microscopy the molecular image of magnetic nanoparticles connected to the stein cells of great biotechnological importance, as well as demonstrating the value of this finding for electronic paramagnetic resonance and its related nanobioscientific value. Ultrastructural results showed the monoclonal antibody anti-CD133 bound to the superparamagnetic nanoparticles by the presence of electrondense granules in cell membrane, as well as in the cytoplasm, revealing the ultrastructural characteristics of CD133(+) cells, exhibiting a round morphology with discrete cytoplasmic projections, having an active nucleus that follows this morphology. The cellular cytoplasm was filled up with mitochondrias, as well as microtubules and vesicles pinocitic. characterizing the process as being related to internalization of the magnetic nanoparticles that were endocyted by the cells in question. Electronic paramagnetic resonance analysis of the CD133(+) stem cells detected that the small (spectrum) generated by the labelled cells comes from the superparamagnetic nanoparticles that are bound to them. These results strongly suggest that these CD133(+) cells can be used in nanobiotechnology applications, with benefits in different biomedical areas.

Quantitative ferromagnetic resonance analysis of CD133 stem cells labeled with iron oxide nanoparticles

The aim of this work is to provide a quantitative method for analysis of the concentration of superparamagnetic iron oxide nanoparticles (SPION), determined by means of ferromagnetic resonance (FMR), with the nanoparticles coupled to a specific antibody (AC133), and thus to express the antigenic labeling evidence for the stem cells C D133(+). The FMR efficiency and sensitivity were proven adequate for detecting and quantifying the low amounts of iron content in the C D133(+) cells (similar to 6.16 x 10(5) pg in the volume of 2 mu l containing 4.5 x 1011 SPION). The quantitative method led to the result of 1.70 x 10(-13) mol of Fe (9.5 pg), or 7.0 x 10(6) nanoparticles per cell. For the quantification analysis via the FMR technique it was necessary to carry out a preliminary quantitative visualization of iron oxide-labeled cells in order to ensure that the nanoparticles coupled to the antibodies are indeed tied to the antigen at the stem cell surface and that the cellular morphology was conserved, as proof of the validity of this method. The quantitative analysis by means of FMR is necessary for determining the signal intensity for the study of molecular imaging by means of magnetic resonance imaging (MRI).

White stem borer damage and grain yield in irrigated rice in West Java, Indonesia

Factors influencing the relationship between whiteheads caused by the white stem borer Scirpophaga innotata (Walker) and grain yield were investigated. We determined the effect of different numbers of whiteheads on grain yield using different cultivars, nitrogen application, and at different field locations in Cilamaya, West Java. At the same number of panicles and whiteheads per plant, yield reduction is greater in cisadane than in IR64. With increasing nitrogen application, the range in panicle height increased. Except for Ketan, more whiteheads were recorded in shorter panicles. Two locations planted to the same cultivar showed different relationships between whiteheads and grain yield. The relationship between whiteheads and grain yield depends on the distribution of whiteheads in the field. Unless these factors have been taken into consideration, it may be difficult to make a damage prediction of white stem borer in the field. (C) 1997 Published by Elsevier Science Ltd.

Limited Ca(2+) and PKA-pathway dependent neurogenic differentiation of human adult mesenchymal stem cells as compared to fetal neuronal stem cells

The ability of mesenchymal stem cells to generate functional neurons in culture is still a matter of controversy. In order to assess this issue, we performed a functional comparison between neuronal differentiation of human MSCs and fetal-derived neural stem cells (NSCs) based on morphological, immunocytochemical, and electrophysiological criteria. Furthermore, possible biochemical mechanisms involved in this process were presented. NF200 immunostaining was used to quantify the yield of differentiated cells after exposure to CAMP. The addition of a PKA inhibitor and Ca(2+) blockers to the differentiation medium significantly reduced the yield of differentiated cells. Activation of CREB was also observed on MSCs during maturation. Na(+)-, K(+)-, and Ca(2+)-voltage-dependent currents were recorded from MSCs-derived cells. In contrast, significantly larger Na(+) currents, firing activity, and spontaneous synaptic currents were recorded from NSCs. Our results indicate that the initial neuronal differentiation of MSCs is induced by CAMP and seems to be dependent upon Ca(2+) and the PKA pathway. However, compared to fetal neural stem cells, adult mesenchymal counterparts are limited in their neurogenic potential. Despite the similar yield of neuronal cells, NSCs achieved a more mature functional state. Description of the underlying mechanisms that govern MSCs` differentiation toward a stable neuronal phenotype and their limitations provides a unique opportunity to enhance our understanding of stem cell plasticity. (C) 2009 Elsevier Inc. All rights reserved.

Stem cells in endometrium and their role in the pathogenesis of endometriosis

The human endometrium is a dynamic tissue that undergoes cycles of growth and regression with each menstrual cycle. Adult progenitor stem cells are likely responsible for this remarkable regenerative capacity; these same progenitor stem cells may also have an enhanced capacity to generate endometriosis if shed in a retrograde fashion. The progenitor stem cells reside in the uterus; however, less-committed mesenchymal stem cells may also travel from other tissues such as bone marrow to repopulate the progenitor population. Mesenchymal stem cells are also involved in the pathogenesis of endometriosis and may be the principle source of endometriosis outside of the peritoneal cavity when they differentiate into endometriosis in ectopic locations. Finally, besides progenitor stem cells, recent publications have identified multipotent stem cells in the endometrium. These multipotent stem cells are a readily available source of cells that are useful in tissue engineering and regenerative medicine. Endometrial stem cells have been used to generate chondrocytes, myocytes, neurons, and adiposites in vitro as well as to replace dopaminergic neurons in a murine model of Parkinson`s disease.

Stem cells in the treatment of chronic spinal cord injury: evaluation of somatosensitive evoked potentials in 39 patients

Study design: A prospective, non-randomized clinical series trial. Objective: To evaluate the effect of autogenous undifferentiated stem cell infusion for the treatment of patients with chronic spinal cord injury (SCI) on somatosensory evoked potentials (SSEPs). Setting: A public tertiary hospital in Sao Paulo, Brazil. Methods: Thirty-nine consecutive patients with diagnosed complete cervical and thoracic SCI for at least 2 years and with no cortical response in the SSEP study of the lower limbs were included in the trial. The trial patients underwent peripheral blood stem cell mobilization and collection. The stem cell concentrate was cryopreserved and reinfused through arteriography into the donor patient. The patients were followed up for 2.5 years and submitted to SSEP studies to evaluate the improvement in SSEPs after undifferentiated cell infusion. Results: Twenty-six (66.7%) patients showed recovery of somatosensory evoked response to peripheral stimuli after 2.5 years of follow-up. Conclusion: The 2.5-year trial protocol proved to be safe and improved SSEPs in patients with complete SCI. Sponsorship: None. Spinal Cord (2009) 47, 733-738; doi: 10.1038/sc.2009.24; published online 31 March 2009

Identification of hepatic stem/progenitor cells in canine hepatocellular and cholangiocellular carcinoma

Hepatic progenitor cells (HPCs) are bipotential stem cells residing in human and animal livers that are able to differentiate towards the hepatocytic or cholangiocytic lineages. HPCs are present in both hepatocellular (HCC) and cholangiocellular carcinoma (CC) in humans; and a small percentage of HCC can originate from cancer stem cells. However, its distribution in canine liver tumour has not been studied. Herein, we searched for stem/progenitor cells in 13 HCC and 7 CC archived samples by immunohistochemical analysis. We found that both liver tumours presented a higher amount of K19-positive HPCs. Besides, 61.6% of HCC cases presented immature CD44-positive hepatocytes. Nevertheless, only two cases presented CD133-positive cells. As observed in humans, hepatic canine tumours presented activated HPCs, with important differentiation onto hepatocytes-like cells and minimal role of cancer stem cells on HCC. These findings reiterate the applicability of canine model in the search for new therapies before application in humans.

Integrating toxicity testing in the wastewater management of chemical storage terminals - A proposal based on a ten-year study

Wastewater control at storage terminals of liquid chemical products in bulk is very difficult because of the variety of products handled in the facilities generating effluents of variable composition. The main objective of this work was to verify if the Vibrio fischeri acute toxicity test could be routinely included in the wastewater management of those facilities along with physical and chemical analysis in order to evaluate and improve the quality of the generated effluents. The study was performed in two phases before and after the implementation of better operational practices/treatment technologies. Chemical oxygen demand (COD) and toxicity of treated effluents did not correlate showing that effluents with low COD contain toxic substances and non-biodegradable organic matter, which may be not degraded when discharged into the aquatic environment. Segregation of influents or pre-treatment based on toxicity results and biodegradability index were implemented in the facilities generating significant improvements in the quality of final effluents with reduction of Biochemical oxygen demand (BOD) and toxicity. The integration of physical and chemical analysis with the V.fischeri toxicity test turned out to be an excellent tool for wastewater management in chemical terminals allowing rapid decision making for pollution control and prevention measures. Reuse of rain water was also proposed and when implemented by the facilities resulted in economical and environmental benefits. (C) 2010 Elsevier B.V. All rights reserved.

Effects of semen storage and separation techniques on sperm DNA fragmentation

Objective: To determine the effect of semen storage and separation techniques on sperm DNA fragmentation. Design: Controlled clinical study. Setting: An assisted reproductive technology laboratory. Patient(s): Thirty normoozospermic semen samples obtained from patients undergoing infertility evaluation. Intervention(s): One aliquot from each sample was immediately prepared (control) for the sperm chromatin dispersion assay (SCD). Aliquots used to assess storage techniques were treated in the following ways: snap frozen by liquid nitrogen immersion, slow frozen with Tris-yolk buffer and glycerol, kept on ice for 24 hours or maintained at room temperature for 4 and 24 hours. Aliquots used to assess separation techniques were processed by the following methods: washed and centrifuged in media, swim-up from washed sperm pellet, density gradient separation, density gradient followed by swim-up. DNA integrity was then measured by SCD. Main Outcome Measure(s): DNA fragmentation as measured by SCD. Result(s): There was no significant difference in fragmentation among the snap frozen, slow frozen, and wet-ice groups. Compared to other storage methods short-term storage at room temperature did not impact DNA fragmentation yet 24 hours storage significantly increased fragmentation. Swim-up, density gradient and density gradient/swim-up had significantly reduced DNA fragmentation levels compared with washed semen. Postincubation, density gradient/swim-up showed the lowest fragmentation levels. Conclusion(s): The effect of sperm processing methods on DNA fragmentation should be considered when selecting storage or separation techniques for clinical use. (Fertil Steril (R) 2010;94:2626-30. (C) 2010 by American Society for Reproductive Medicine.)

Pleural fluid: Are temperature and storage time critical preanalytical error factors in biochemical analyses?

Background: Biochemical analysis of fluid is the primary laboratory approach hi pleural effusion diagnosis. Standardization of the steps between collection and laboratorial analyses are fundamental to maintain the quality of the results. We evaluated the influence of temperature and storage time on sample stability. Methods: Pleural fluid from 30 patients was submitted to analyses of proteins, albumin, lactic dehydrogenase (LDH), cholesterol, triglycerides, and glucose. Aliquots were stored at 21 degrees, 4 degrees, and-20 degrees C, and concentrations were determined after 1, 2, 3, 4, 7, and 14 days. LDH isoenzymes were quantified in 7 random samples. Results: Due to the instability of isoenzymes 4 and 5, a decrease in LDH was observed in the first 24 h in samples maintained at -20 degrees C and after 2 days when maintained at 4 degrees C. Aside from glucose, all parameters were stable for up to at least day 4 when stored at room temperature or 4 degrees C. Conclusions: Temperature and storage time are potential preanalytical errors in pleural fluid analyses, mainly if we consider the instability of glucose and LDH. The ideal procedure is to execute all the tests immediately after collection. However, most of the tests can be done in refrigerated sample;, excepting LDH analysis. (C) 2010 Elsevier B.V. All rights reserved.

Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered for human regenerative therapy applications, and safe culture and expansion protocols are needed especially in the context of interspecies contamination. Human platelet lysate (PL) has been proposed as animal serum substitute during in vitro MSC expansion. In this work, a simplified and efficient method to obtain autologous PL to replace animal serum in cell culture applications is described. STUDY DESIGN AND METHODS: PL obtained by freezing and centrifugation procedures was tested as medium supplement for human adipose mesenchymal stem cell (hASC) culture. Differential proliferation, immunophenotypic changes, and differentiation under PL or fetal bovine serum (FBS) were assessed. RESULTS: In contrast to 10% FBS supplementation, cell population doubling time was significantly lower when hASCs were cultured with the same concentration of PL ( PL 22.9 +/- 1.5 hr vs. FBS 106.7 +/- 6.5 hr, t test, p < 0.05). Furthermore, hASCs maintained with 2.5% PL supplementation also showed satisfactory results. Immunophenotypic analysis revealed no differences between hASCs cultivated with PL or FBS supplementation and both cultures retained the potential to differentiate into adipose cells. These results demonstrate that autologous PL obtained from the same donor can be used as animal serum substitute in hASC culture. CONCLUSIONS: Taken together, evidence is provided that platelets provided by a single donor are sufficient to obtain PL for hASC propagation for clinical-scale applications mitigating the potential untoward side effects associated with the use of animal-derived reagents.

Molecular Characterization of Strains of Respiratory Syncytial Virus Identified in a Hematopoietic Stem Cell Transplant Outpatient Unit Over 2 Years: Community or Nosocomial Infection?

Respiratory syncytial virus (RSV) is recognized as the leading cause of nosocomial respiratory infection among hematopoietic stem cell transplant (HSCT) recipients, causing considerable morbidity and mortality. RSV is easily transmitted by contact with contaminated surfaces, and in HSCT units, more than 50% of RSV infections have been characterized as of nosocomial origin. From April 2001 to October 2002, RSV was identified by direct immunofluorescent assay in 42 symptomatic HSCT recipients. Seven RSV strains from 2001 and 12 RSV strains from 2002 were sequenced. RNA extraction, cDNA synthesis, and seminested polymerase chain reaction (PCR) with primers complementary to RSV genes G and F were pet-formed. PCR products were analyzed by nucleotide sequencing of the C-terminal region of gene G for typing (in group A or B). Of the 7 strains analyzed in 2001, only 2 belonged to group B; the other 5 belonged to group A. Of these 7 strains, 3 were identical and were from recipients receiving outpatient care. In 2002, of the 12 strains analyzed, 3 belonged to group A and the other 9 belonged to group B. Of these 9 strains, 7 were genetically identical and were also from recipients receiving outpatient care. Therefore, multiple strains of RSV cocirculated in the hematopoietic stem cell transplant units (ward and outpatient units) between 2001 and 2002. Nosocomial transmission was more likely to occur at the HSCT outpatient unit than in the HSCT ward. Infection control practices should also be implemented in the outpatient setting.

Mesenchymal Stem Cell Properties of Periodontal Ligament Cells From Deciduous and Permanent Teeth

Background: Human postnatal stem cells have been identified in periodontal ligaments (PDLs). In this study, the in vitro biologic properties of CD105(+) enriched cell subsets from PDLs harvested from deciduous (DePDL) and permanent (PePDL) teeth are comparatively assessed. Methods: PDL tissue was obtained from 12 teeth (six primary and six permanent) from which CD105(+) CD34(-) CD45(-) cells were isolated by magnetic cell sorting. To identify and quantitatively compare the stem cell markers, DePDL and PePDL cells were assessed for CD166 surface antigen expression by flow cytometry, real-time polymerase chain reaction, and immunostaining for Stro-1 and Oct-4, osteogenic and adipogenic differentiation, and proliferation rate by trypan blue method. Results: Magnetic cell sorting isolated cell populations containing 23.87% (+/- 11.98%) and 11.68% (+/- 6.27%) of CD105(+) expressing cells from PePDL and DePDL, respectively. Flow cytometric analysis demonstrated a higher proportion of CD105(+) cells coexpressing CD166 surface antigen in PePDL, whereas immunostaining and real-time polymerase chain reaction analysis demonstrated that both cell subsets expressed Stro-1 and Oct-4. DePDL-CD105(+) subsets were more proliferative compared to PePDL subsets, and both cell populations showed multipotential capabilities to differentiate in vitro to osteoblast/cementoblast- and adipocyte-like cells. However, a higher expression of adipogenic-related genes was observed in DePDL cells, whereas PePDL-CD105(+) cell subset presented a more homogeneous osteoblast/cementoblast response. Conclusion: These findings demonstrate that highly purified mesenchymal progenitor cell subsets can be obtained from the PDLs of both deciduous and permanent teeth, and further indicate phenotype dissimilarities that may have an impact on their clinical applications. J Periodontol 2010;81:1207-1215.