1,721,001 research outputs found
Is stem cell chromosomal stability affected by cryopreservation conditions?
No full textThe use of in vitro cultured stem cells in cell therapy, must meet at least two requirements: the preservation of a normal genetic karyotype and maintenance of integrity during long term-culturing and storage in liquid nitrogen tanks. Here we report on the karyological characterization of neural stem cells derived from adult (ANS) subentricular zone (SVZ), mouse fetal cotex cells, mESCs cells NS46 (LC-1) and related engineered clones as well as Neurospheres from the mouse striatum Li-4. The results show a series of chromosomal aberrations present in these cells in agreement with previous reports. On the other hand, karyotyping of the human neural stem cell lines derived from hESCs (H9-hNCPC) and fetal cerebellum (CB541) showed a stable genomic asset with few chromosomal aberration. These results underscore the importance of performing routine cytogenetic analysis before using the cells to produce chimeric mice or in studies of lineage specific differentiation. We are also evaluating the use of Cell microarray for high-throughput screening of stem cell differentiation by immunocytochemical analysis. With this technique we were able to follow the expression of neural differentiation markers and the involvement of SEL1L gene in stem cell self-renewal
Low-flow ischemia and hypoxia stimulate apoptosis in perfused rat hearts independently of reperfusion
Full text linkPost-ischemic reperfusion leads to apoptosis-linked loss of myocytes in cultured cells and in vivo. We tested the hypothesis that apoptosis develops without reperfusion in Langendorff-perfused hearts exposed to either low-flow ischemia (LFI) or hypoxia (H). Rat hearts were perfused with aminoacid-enriched Krebs- Henseleit buffer and exposed for 6 h to LFI (flow=2 ml/min, PO2=500±50mmHg, mean±SD), H (10ml/min, 120±15mmHg), or control conditions (C, 10ml/min, 500±50mmHg). At selected times, DNA-fragmentation was measured by agarose-gel electrophoresis and in situ TUNEL assay. After 6 h, the ratio (TUNEL-positi- ve)/(total nuclei) was 0.620±0.027, 0.615±0.005, 0.404±0.021 in LFI, H and C, respectively. The ratio was 0.813±0.021 in hearts exposed to 90 min global no-flow ischemia and reperfused (5 h). To assess the role of membrane-diffusible factors, separate experiments were performed recirculating the medium and exposing hearts to LFI or H as above. The degree of apoptosis was the same in both the recirculating
and non-recirculating modes. Thus, apoptosis develops by similar extents and in a time-dependent fashion in crystalloid-perfused rat hearts during LFI or H at the same oxygen shortage (flow•PO2), even without the reperfusion
Evidence of apoptosis in the crystalloid-perfused isolated rat heart during low-flow ischemia or hypoxia
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Analysis of protein ions in the range 3000-12000 Th under partial (no discharge) atmospheric pressure chemical ionization conditions using ion trap mass spectrometry
No full textA new approach, based on the use of atmospheric pressure chemical ionization ion
trap mass spectrometry (APCI-ITMS), but without a corona discharge, was
investigated for application to creating and monitoring protein ions. It must be
emphasized that APCI is not usually used in protein analysis. In order to verify
the applicability of the proposed method to the analysis of proteins, two
standard proteins (horse cytochrome c and horse myoglobin) were analyzed. A
mixture of the two proteins was also analyzed showing that this novel approach,
based on the use of APCI, can be used in the analysis of protein mixtures
iPS cells : quality controls and validation to become a cell product
No full textHuman embryonic stem cells (hESCs) provide new resources for the biomedical community by virtue of their dual capacities: long-term self-renewal and production of differentiated progeny. hESCs may constitute an endless source of cells for the treatment of debilitating diseases and degenerative disorders. In the last few years alternatives to hESCs have been generated, the ”induced pluripotent stem cells” (iPS) which can repro-gram their adult somatic fate to acquire an embryonic-like feature thanks to the ectopic over-expression of four transcription factors (OCT-4, SOX-2, KL44 and c-MYC). iPS cells nowadays represent the biggest expectation for the emerging molecular stem cell medicine branch, although they are not as versatile as the hESCs, but much still needs to be learned of their characteristics and capacities. These cells retain their own genome and an imprint or un-erasable memory of their somatic cellular origin and whatever has occurred in the organ from which they originate. It is then of paramount importance to invest on “methyloma” or methylation profiles of these cells in order to identify their memory stick.
Since the number of iPS lines will steadily increase, to become an accreditable cell product, they must be submitted to a number of quality control tests to assure that they are morphologically and biochemically undistinguishable from ESCs. Particularly it is essential to analyze the reactivation of the appropriate stage-specific embryonic antigens, the capacity to differentiate into lineages from all three embryonic germ layers, the teratomas formation in immunodeficient mice, the expression of functional telomerase, the epigenetic status and the transcriptionally permissive chromatin structure of pluripotent genes. iPS should be evaluated by integrative genome-wide approaches, allowing the supervising of pluripotency, or the eventual DNA damage subsequent to the reprogramming
SEL1L and squamous cell carcinoma of the esophagus
No full textThe gene SEL1L is involved both in human breast and pancreatic cancer progression. It is located on 14q24.3-31, a region known to be lost in invasive cancer of the esophagus. We aimed to assess whether SEL1L could become a useful biomarker for this cancer. We assessed SEL1L mRNA and protein expression in 35 patients and found it to be weak in low-grade and strong in high-grade dysplasia. Although the majority of cancer patients showed differential expression (mRNA and protein) of SEL1L, in five cases it was completely absent; these patients had the worst outcomes. SEL1L immunoreactivity was negative in normal tissue samples from five patients with mild esophagitis as well as in normal mucosa adjacent to the tumor. We hypothesize that SEL1L could influence those cellular changes that mediate the transition from a normal mucosa to a neoplastic lesion and may help in the identification of those patients at higher risk of developing this cancer. The specific impact of SEL1L in esophageal cancer needs further investigation
Analysis of peptides using partial (no discharge) atmospheric pressure chemical ionization conditions with ion trap mass spectrometry
No full textA novel approach, based on the use of atmospheric pressure chemical ionization
ion trap mass spectrometry (APCI-ITMS) conditions, but without using corona
discharge, was used to analyze peptides. The proposed method was applied to
three standard peptides (bombesin, trityrosine and tyrosine-glycine-glycine) as
well as peptides obtained through enzymatic digestion of two standard proteins
(horse cytochrome c and horse myoglobin)
Cell and Tissue Microarray Technologies for Protein and Nucleic Acid Expression Profiling
No full textTissue microarray (TMA) and cell microarray (CMA) are two powerful techniques that allow for the immunophenotypical characterization of hundreds of samples simultaneously. In particular, the CMA approach is particularly useful for immunophenotyping new stem cell lines (e.g., cardiac, neural, mesenchymal) using conventional markers, as well as for testing the specificity and the efficacy of newly developed antibodies. We propose the use of a tissue arrayer not only to perform protein expression profiling by immunohistochemistry but also to carry out molecular genetics studies. In fact, starting with several tissues or cell lines, it is possible to obtain the complete signature of each sample, describing the protein, mRNA and microRNA expression, and DNA mutations, or eventually to analyze the epigenetic processes that control protein regulation. Here we show the results obtained using the Galileo CK4500 TMA platform
Tissue microarrays : a cross-over validation tool for research
No full textBackground: Genomic, transcriptomic, proteomic have increased the importance of tissues in the discovery and validation of prognostic, diagnostic and therapeutic biomarkers. Human tissue has become a strategic and unavoidable tool for all –omics technologies including high-throughput in situ proteomics to study signal transduction and protein-protein interactions. Tissue microarray (TMA) systems allow the study of hundreds of samples (paraffin embedded tissues or cells) on a single slide for hystochemical analysis. The “Galileo CK4500” (www.isenet.it) is a high-throughput semiautomatic and computer-assisted TMA platform with the unique feature of picking cores of interest in a tissue or cell block and extract nucleic acids for subsequent molecular biology analysis.
Methods: Harvested and agarose re-suspended Neural Stem Cells (NSCs) were fixed in PFA (4%) and treated like the human glioblastoma xenografts tissue donor block selected for the specific study. Cores of cells or tissues (previously selected) from the donor blocks were placed by the arrayer either in a second paraffin block or in appropriate vessels to be processed for nucleic acid extraction.
Results: TMA and CLMA (Cell Line Microarray) technology were used to analyse glioblastoma xenografts and NSCs, to verify stemness and tripotency. From the cores DNA, RNA and microRNAs were successfully extracted.
Conclusions: The Galileo CK4500 platform is a powerful pathology tool to simultaneously screen a huge number of tissues or cell lines treated in different conditions or with different phenotypes. This technique will be particularly useful to obtain immunophenotypical information and to perform epigenetic studies in specific selected areas of a biological sample
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