Cell fusion is an important technique that is used in the field of medicine and biomedical research. For instance, fusion can be used to create hybridomas and novel types of secretory hybrid cells. It may also be used to engineer cultured insulin-secreting pancreatic B-cell lines for the treatment of diabetes. Historically, the applications listed above have been accomplished by a number of methodologies including dielectrophoresis, centrifugation, polyethylene glycol (PEG) and viral fusion proteins. However, these approaches often fail to produce the desired results due to poor cell viability, lack of 1:1 fusion, and use of non-physiological environments. It is proposed that the application of an electrical field generated by corona charge (corona fields) and subsequent treatment with direct current (DC) pulse technology will overcome these deficiencies. Isolated and pre-labeled neuronally committed human teratocarcinoma (NT2) cells in monoculture or co-culture, were seeded in chambers, constructed in the laboratory, and allowed to adhere to the chamber bottom prior to corona treatment. A corona generator, also constructed in the laboratory, was used to expose cells to positive and negative electrical charges to induce cell-cell contact. The cells were then pulsed with DC voltage to induce fusion. During the experiments, cells were photographed sequentially to record cell movement/contact and fusion. The project was designed to identify optimal corona-based electrofusion parameters for viable, 1:1 cell fusion. Optimal results for cell-cell contact were obtained using a cell density of 2.35 times ten to the fourth power cells per microliter Dulbecco's Modified Eagle Medium (DMEM) in a grounded circular plate corona chamber following at least 3 minutes of settling time. Corona charges from (+) 6.1 kilivolt and (-) 5.5 kilivolt potentials were determined as being most favorable for cell movement and viability. Fusion was best achieved by first exposing either a circular or square ungrounded corona chamber configuration to 3 minutes (+) corona charge followed by 3 minutes (--) corona charge; disturbing the cells in the chamber with mechanical force; and then exposing them to 8-15 sequences of a 2,500 Volts per centimeter DC pulse at 100 microseconds.

Introduction Adipocytes are part of hematopoietic microenvironment, even though they are generally believed to play no active role during hematopoiesis. We have shown that accumulation of fat cells in femoral bone marrow (BM) coincides with increased expression of neuropilin-1 (NP-1), while it is weakly expressed in hematopoietic iliac crest BM. Starting from this observation, we postulated that adipocytes might exert a negative effect on hematopoiesis mediated through NP-1. Material and Methode To test this hypothesis, we set up BM adipocytes differentiated into fibroblast-like fat cells (FLFC), which share the major characteristics of primitive unilocular fat cells, as an experimental model. Results Morphological and immunophenotypic analysis of FLFCs in co-culture with CD34+ cells revealed that FLFCs constitutively produced M-CSF and induced CD34+ differentiation into macrophages independently of cell-to-cell contact. By contrast, granulopoiesis was hampered by cell-to-cell contact but could be restored in transwell culture conditions, together with G-CSF production. Both functions were also recovered when FLFCs cultured in contact with CD34+ cells were treated with an antibody neutralizing NP-1, which proved its critical implication in contact inhibition. Conclusion Our data provide the first evidence that adipocytes exert regulatory functions during hematopoiesis that might be implicated in some pathological processes.

Indiana University-Purdue University Indianapolis (IUPUI) / Approximately 2.8% of the world population is currently infected with hepatitis C virus (HCV). Neutralizing antibodies (nAbs) are often generated in chronic hepatitis C patients yet fail to control the infection. In the first two chapters of this study, we focused on two alternative routes of HCV transmission, which may contribute to HCV’s immune evasion and establishment of chronic infection. HCV was transmitted via a cell-cell contact-mediated (CCCM) route and in the form of exosomes. Formation of HCV infection foci resulted from CCCM HCV transfer and was cell density-dependent. Moreover, CCCM HCV transfer occurred rapidly, involved all four known HCV receptors and intact actin cytoskeleton, and led to productive HCV infection. Furthermore, live cell imaging revealed the temporal and spatial details of the transfer process. Lastly, HCV from HCV-infected hepatocytes and patient plasma occurred in both exosome-free and exosome-associated forms and the exosome-associated HCV remained infectious, even though HCV infection did not significantly alter exosome secretion. In the third chapter, we characterized HCV interaction with astrocytes, one of the putative HCV target cells in the brain. HCV infection causes the central nervous system (CNS) abnormalities in more than 50% of chronically infected subjects but the underlying mechanisms are largely unknown. We showed that primary human astrocytes (PHA) were very inefficiently infected by HCV, either in the free virus form or through cell-cell contact. PHA expressed all known HCV receptors but failed to support HCV entry. HCV IRES-mediated translation was functional in PHA and further enhanced by miR122 expression. Nevertheless, PHA did not support HCV replication regardless of miR122 expression. To our great surprise, HCV exposure induced robust IL-18 expression in PHA and exhibited direct neurotoxicity. In summary, we showed that CCCM HCV transfer and exosome-mediated HCV infection constituted important routes for HCV infection and dissemination and that astrocytes did not support productive HCV infection and replication, but HCV interactions with astrocytes and neurons alone might be sufficient to cause CNS dysfunction. These findings provide new insights into HCV infection of hepatocytes and astrocytes and shall aid in the development of new and effective strategies for preventing and treating HCV infection.