288 research outputs found

    The ImageStream System: a key step to a new era in imaging.

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    The aim of this article is to provide a brief review about the ImageStream system a novel tool for multiparameter cell analysis in flow. The instrument integrates the features of flow cytometry and fluorescence microscopy combined with a modern methodology for image analysis. Similar to flow cytometry, ImageStream allows analysis of a large number of cells based on their fluorescence features and provides statistical analysis of these features. Additionally, ImageStream allows detailed morphometric cellular analysis based on acquired cellular images integrating various morphometric and photometric features of the examined cells. Simply stated, ImageStream system is an advanced flow cytometer acquiring both integrated fluorescence signals as well as high quality fluorescence images and allowing muliparameter analysis. The innovative features of the instrument offer new analytical capabilities and allow for a multitude of possible applications beyond the current means of flow cytometry. While this article summarizes basic information about the features of ImageStream and its applications based on the available literature and it also describes our own experience

    Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts

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    Fast and efficient homing and engraftment of hematopoietic stem progenitor cells (HSPCs) is crucial for positive clinical outcomes from transplantation. We found that this process depends on activation of the Nlrp3 inflammasome, both in the HSPCs to be transplanted and in the cells in the recipient bone marrow (BM) microenvironment. For the first time we provide evidence that functional deficiency in the Nlrp3 inflammasome in transplanted cells or in the host microenvironment leads to defective homing and engraftment. At the molecular level, functional deficiency of the Nlrp3 inflammasome in HSPCs leads to their defective migration in response to the major BM homing chemoattractant stromal-derived factor 1 (SDF-1) and to other supportive chemoattractants, including sphingosine-1-phosphate (S1P) and extracellular adenosine triphosphate (eATP). We report that activation of the Nlrp3 inflammasome increases autocrine release of eATP, which promotes incorporation of the CXCR4 receptor into membrane lipid rafts at the leading surface of migrating cells. On the other hand, a lack of Nlrp3 inflammasome expression in BM conditioned for transplantation leads to a decrease in expression of SDF-1 and danger-associated molecular pattern molecules (DAMPs), which are responsible for activation of the complement cascade (ComC), which in turn facilitates the homing and engraftment of HSPCs

    Very small embryonic-like stem cells (VSELs) represent a real challenge in stem cell biology : recent pros and cons in the midst of a lively debate

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    The concept that adult tissue, including bone marrow (BM), contains early-development cells with broader differentiation potential has again been recently challenged. In response, we would like to review the accumulated evidence from several independent laboratories that adult tissues, including BM, harbor a population of very rare stem cells that may cross germ layers in their differentiation potential. Thus, the BM stem cell compartment hierarchy needs to be revisited. These dormant, early-development cells that our group described as very small embryonic-like stem cells (VSELs) most likely overlap with similar populations of stem cells that have been identified in adult tissues by other investigators as the result of various experimental strategies and have been given various names. As reported, murine VSELs have some pluripotent stem cell characteristics. Moreover, they display several epiblast/germline markers that suggest their embryonic origin and developmental deposition in adult BM. Moreover, at the molecular level, changes in expression of parentally imprinted genes (for example, Igf2–H19) and resistance to insulin/insulin-like growth factor signaling (IIS) regulates their quiescent state in adult tissues. In several emergency situations related to organ damage, VSELs can be activated and mobilized into peripheral blood, and in appropriate animal models they contribute to tissue organ/regeneration. Interestingly, their number correlates with lifespan in mice, and they may also be involved in some malignancies. VSELs have been successfully isolated in several laboratories; however, some investigators experience problems with their isolation

    Very Small Embryonic-Like Stem Cells Purified from Umbilical Cord Blood Lack Stem Cell Characteristics

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    Very small embryonic-like (VSEL) cells have been described as putatively pluripotent stem cells present in murine bone marrow and human umbilical cord blood (hUCB) and as such are of high potential interest for regenerative medicine. However, there remain some questions concerning the precise identity and properties of VSEL cells, particularly those derived from hUCB. For this reason, we have carried out an extensive characterisation of purified populations of VSEL cells from a large number of UCB samples. Consistent with a previous report, we find that VSEL cells are CXCR4+, have a high density, are indeed significantly smaller than HSC and have an extremely high nuclear/cytoplasmic ratio. Their nucleoplasm is unstructured and stains strongly with Hoechst 33342. A comprehensive FACS screen for surface markers characteristic of embryonic, mesenchymal, neuronal or hematopoietic stem cells revealed negligible expression on VSEL cells. These cells failed to expand in vitro under a wide range of culture conditions known to support embryonic or adult stem cell types and a microarray analysis revealed the transcriptional profile of VSEL cells to be clearly distinct both from well-defined populations of pluripotent and adult stem cells and from the mature hematopoietic lineages. Finally, we detected an aneuploid karyotype in the majority of purified VSEL cells by fluorescence in situ hybridisation. These data support neither an embryonic nor an adult stem cell like phenotype, suggesting rather that hUCB VSEL cells are an aberrant and inactive population that is not comparable to murine VSEL cells

    Differentiation of human multipotent dermal fibroblasts into islet-like cell clusters

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    <p>Abstract</p> <p>Background</p> <p>We have previously obtained a clonal population of cells from human foreskin that is able to differentiate into mesodermal, ectodermal and endodermal progenies. It is of great interest to know whether these cells could be further differentiated into functional insulin-producing cells.</p> <p>Results</p> <p>Sixty-one single-cell-derived dermal fibroblast clones were established from human foreskin by limiting dilution culture. Of these, two clones could be differentiated into neuron-, adipocyte- or hepatocyte-like cells under certain culture conditions. In addition, those two clones were able to differentiate into islet-like clusters under pancreatic induction. Insulin, glucagon and somatostatin were detectable at the mRNA and protein levels after induction. Moreover, the islet-like clusters could release insulin in response to glucose in vitro.</p> <p>Conclusions</p> <p>This is the first study to demonstrate that dermal fibroblasts can differentiate into insulin-producing cells without genetic manipulation. This may offer a safer cell source for future stem cell-based therapies.</p

    CXCR4 expression on circulating pan-cytokeratin positive cells is associated with survival in patients with advanced non-small cell lung cancer

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    <p>Abstract</p> <p>Background</p> <p>The CXC chemokine, CXCL12, and its receptor, CXCR4 promote metastases of a variety of solid tumors, including non-small cell lung cancer (NSCLC). The expression of CXCR4 on tumor cells may represent a critical biomarker for their propensity to metastasize. This study was performed to evaluate the hypothesis that co-expression of pan-cytokeratin and CXCR4 may be a prognostic marker for patients with advanced NSCLC.</p> <p>Methods</p> <p>We evaluated CXCR4 levels on circulating pan-cytokeratin positive cells from patients with NSCLC. NSCLC tumor and metastases were also assessed for the presence of CXCR4.</p> <p>Results</p> <p>Pan-cytokeratin positive cells were increased in the circulation of patients with NSCLC, as compared to normal control subjects. Patients with pan-cytokeratin +/CXCR4+ = 2,500 cells/ml had a significant improvement in median survival when compared with patients with pan-cytokeratin +/CXCR4+ >2,500 cells/ml (not achieved versus 14 weeks). CXCR4 expression was found on NSCLC tumors and at sites of tumor metastasis.</p> <p>Conclusion</p> <p>This study suggests that CXCR4 may be a prognostic marker in NSCLC, and provides hypothesis-generating results, which may be important in determining metastatic potential. In future studies, we will prospectively evaluate the prognostic significance of pan-cytokeratin/CXCR4+ cells, and determine the mechanisms involved in the regulation of CXCR4 expression on tumor cells in a larger patient population.</p

    Effect of chemokine receptor CXCR4 on hypoxia-induced pulmonary hypertension and vascular remodeling in rats

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    <p>Abstract</p> <p>Background</p> <p>CXCR4 is the receptor for chemokine CXCL12 and reportedly plays an important role in systemic vascular repair and remodeling, but the role of CXCR4 in development of pulmonary hypertension and vascular remodeling has not been fully understood.</p> <p>Methods</p> <p>In this study we investigated the role of CXCR4 in the development of pulmonary hypertension and vascular remodeling by using a CXCR4 inhibitor AMD3100 and by electroporation of CXCR4 shRNA into bone marrow cells and then transplantation of the bone marrow cells into rats.</p> <p>Results</p> <p>We found that the CXCR4 inhibitor significantly decreased chronic hypoxia-induced pulmonary hypertension and vascular remodeling in rats and, most importantly, we found that the rats that were transplanted with the bone marrow cells electroporated with CXCR4 shRNA had significantly lower mean pulmonary pressure (mPAP), ratio of right ventricular weight to left ventricular plus septal weight (RV/(LV+S)) and wall thickness of pulmonary artery induced by chronic hypoxia as compared with control rats.</p> <p>Conclusions</p> <p>The hypothesis that CXCR4 is critical in hypoxic pulmonary hypertension in rats has been demonstrated. The present study not only has shown an inhibitory effect caused by systemic inhibition of CXCR4 activity on pulmonary hypertension, but more importantly also has revealed that specific inhibition of the CXCR4 in bone marrow cells can reduce pulmonary hypertension and vascular remodeling via decreasing bone marrow derived cell recruitment to the lung in hypoxia. This study suggests a novel therapeutic approach for pulmonary hypertension by inhibiting bone marrow derived cell recruitment.</p

    CXC receptor-4 mRNA silencing abrogates CXCL12-induced migration of colorectal cancer cells

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    <p>Abstract</p> <p>Background</p> <p>Interactions between CXCR4 and its ligand CXCL12 have been shown to be involved in cancer progression in colorectal cancer (CRC). We performed a comparative CXCL12/CXCR4 expression analysis and assessed the effect of external CXCL12 stimulation on migration of CRC cells without and with CXCR4 inhibition.</p> <p>Methods</p> <p>Expression of CXCL12/CXCR4 was assessed by quantitative real-time PCR, ELISA and immunohistochemistry in resection specimens of 50 CRC patients as well as in the corresponding normal tissues and in three human CRC cell lines with different metastatic potential (Caco-2, SW480 and HT-29). Migration assays were performed after stimulation with CXCL12 and CXCR4 was inhibited by siRNA and neutralizing antibodies.</p> <p>Results</p> <p>In CRC tissues CXCL12 was significantly down-regulated and CXCR4 was significantly up-regulated compared to the corresponding normal tissues. In cell lines CXCR4 was predominantly expressed in SW480 and less pronounced in HT-29 cells. CXCL12 was only detectable in Caco-2 cells. CXCL12 stimulation had no impact on Caco-2 cells but significantly increased migration of CXCR4 bearing SW480 and HT-29 cells. This effect was significantly abrogated by neutralizing anti-CXCR4 antibody as well as by CXCR4 siRNAs (P < 0.05).</p> <p>Conclusions</p> <p>CXCR4 expression was up-regulated in CRC and CXCL12 stimulation increased migration in CXCR4 bearing cell lines. Migration was inhibited by both neutralizing CXCR4 antibodies and CXCR4 siRNAs. Thus, the expression and functionality of CXCR4 might be associated with the metastatic potential of CRC cells and CXCL12/CXCR4 interactions might therefore constitute a promising target for specific treatment interventions.</p

    Investigation of Cellular and Molecular Responses to Pulsed Focused Ultrasound in a Mouse Model

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    Continuous focused ultrasound (cFUS) has been widely used for thermal ablation of tissues, relying on continuous exposures to generate temperatures necessary to induce coagulative necrosis. Pulsed FUS (pFUS) employs non-continuous exposures that lower the rate of energy deposition and allow cooling to occur between pulses, thereby minimizing thermal effects and emphasizing effects created by non-thermal mechanisms of FUS (i.e., acoustic radiation forces and acoustic cavitation). pFUS has shown promise for a variety of applications including drug and nanoparticle delivery; however, little is understood about the effects these exposures have on tissue, especially with regard to cellular pro-homing factors (growth factors, cytokines, and cell adhesion molecules). We examined changes in murine hamstring muscle following pFUS or cFUS and demonstrate that pFUS, unlike cFUS, has little effect on the histological integrity of muscle and does not induce cell death. Infiltration of macrophages was observed 3 and 8 days following pFUS or cFUS exposures. pFUS increased expression of several cytokines (e.g., IL-1α, IL-1β, TNFα, INFγ, MIP-1α, MCP-1, and GMCSF) creating a local cytokine gradient on days 0 and 1 post-pFUS that returns to baseline levels by day 3 post-pFUS. pFUS exposures induced upregulation of other signaling molecules (e.g., VEGF, FGF, PlGF, HGF, and SDF-1α) and cell adhesion molecules (e.g., ICAM-1 and VCAM-1) on muscle vasculature. The observed molecular changes in muscle following pFUS may be utilized to target cellular therapies by increasing homing to areas of pathology
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