Osteoclast Fusion: Time‐Lapse Reveals Involvement of CD47 and Syncytin‐1 at Different Stages of Nuclearity

Investigations addressing the molecular keys of osteoclast fusion are primarily based on end‐point analyses. No matter if investigations are performed in vivo or in vitro the impact of a given factor is predominantly analyzed by counting the number of multi‐nucleated cells, the number of nuclei per multinucleated cell or TRAcP activity. But end‐point analyses do not show how the fusion came about. This would not be a problem if fusion of osteoclasts was a random process and occurred by the same molecular mechanism from beginning to end. However, we and others have in the recent period published data suggesting that fusion partners may specifically select each other and that heterogeneity between the partners seems to play a role. Therefore, we set out to directly test the hypothesis that fusion factors have a heterogenic involvement at different stages of nuclearity. Therefore, we have analyzed individual fusion events using time‐lapse and antagonists of CD47 and syncytin‐1. All time‐lapse recordings have been studied by two independent observers. A total of 1808 fusion events were analyzed. The present study shows that CD47 and syncytin‐1 have different roles in osteoclast fusion depending on the nuclearity of fusion partners. While CD47 promotes cell fusions involving mono‐nucleated pre‐osteoclasts, syncytin‐1 promotes fusion of two multi‐nucleated osteoclasts, but also reduces the number of fusions between mono‐nucleated pre‐osteoclasts. Furthermore, CD47 seems to mediate fusion mostly through broad contact surfaces between the partners’ cell membrane while syncytin‐1 mediate fusion through phagocytic‐cup like structure. J. Cell. Physiol. 232: 1396–1403, 2017. © 2016 Wiley Periodicals, Inc.     

The construction of viable nuclear-cytoplasmic hybrid cells by nuclear transplantation.

Using the mouse L-cell line as a model system, a generalized approach is presented for nuclear transplantation in cultured cells resulting in the construction of cytoplasmic-nuclear hybrid cells. Techniques were developed for the preparation of cytoplast and karyoplasts having minimum contamination by parent whole cells. Sendai virusmediated fusion was performed in a manner which maximized the formation of the desired fusion products-cells having one cell equivalent of cytoplasm from one parent and a nucleus from a second parent. The viability of the fusion products was established by examination of photographic records of the developing cultures. Using these techniques, we found that nuclei could be introduced routinely into 10-30% of a cytoplast culture. From determinations of the increase in cell number with time, it was estimated that at least 30% of the reconstructed cells were capable of division. The approach was next applied to the formation of hybrid cells from L-cell cytoplasts and A9 cell karyoplasts. The A9 cell line is an azaguanine-resistant derivative of L cells. Thus any whole cells remaining in the culture of fused cells were readily eliminated by treatment with the purine analogue. The culture of remaining cytoplasmic-nuclear hybrid cells grew to confluence in the presence of azaguanine. The applicability of the approach to the construction of hybrid cells using parent lines from different organisms is briefly discussed.     

Adipose-derived stem cells and cancer cells fuse to generate cancer stem cell-like cells with increased tumorigenicity

Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cells isolated from adipose tissue and have the ability to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Despite their great therapeutic potentials, previous studies showed that ADSCs could enhance the proliferation and metastatic potential of breast cancer cells (BCCs). In this study, we found that ADSCs fused with BCCs spontaneously, while breast cancer stem cell (CSC) markers CD44⁺CD24^-/lowEpCAM⁺ were enriched in this fusion population. We further assessed the fusion hybrid by multicolor DNA FISH and mouse xenograft assays. Only single nucleus was observed in the fusion hybrid, confirming that it was a synkaryon. In vivo mouse xenograft assay indicated that the tumorigenic potential of the fusion hybrid was significantly higher than that of the parent tumorigenic triple-negative BCC line MDA-MB-231. We had compared the fusion efficiency between two BCC lines, the CD44-rich MDA-MB-231 and the CD44-poor MCF-7, with ADSCs. Interestingly, we found that the fusion efficiency was much higher between MDA-MB-231 and ADSCs, suggesting that a potential mechanism of cell fusion may lie in the dissimilarity between these two cell lines. The cell fusion efficiency was hampered by knocking down the CD44. Altogether, our findings suggest that CD44-mediated cell fusion could be a potential mechanism for generating CSCs.     

Myogenic differentiation of mesenchymal stem cells co‐cultured with primary myoblasts

TE (tissue engineering) of skeletal muscle is a promising method to reconstruct loss of muscle tissue. This study evaluates MSCs (mesenchymal stem cells) as new cell source for this application. As a new approach to differentiate the MSCs towards the myogenic lineage, co‐cultivation with primary myoblasts has been developed and the myogenic potential of GFP (green fluorescent protein)‐transduced rat MSC co‐cultured with primary rat myoblasts was assessed by ICC (immunocytochemistry). Myogenic potential of MSC was analysed by ICC, FACS and qPCR (quantitative PCR). MSC—myoblast fusion phenomena leading to hybrid myotubes were evaluated using a novel method to evaluate myotube fusion ratios based on phase contrast and fluorescence microscopy. Furthermore, MSC constitutively expressed the myogenic markers MEF2 (myogenic enhancer factor 2) and α‐sarcomeric actin, and MEF2 expression was up‐regulated upon co‐cultivation with primary myoblasts and the addition of myogenic medium supplements. Significantly higher numbers of MSC nuclei were involved in myotube formations when bFGF (basic fibroblast growth factor) and dexamethasone were added to co‐cultures. In summary, we have determined optimal co‐culture conditions for MSC myogenic differentiation up to myotube formations as a promising step towards applicability of MSC as a cell source for skeletal muscle TE as well as other muscle cell‐based therapies.     

The Problem of Colliding Networks and its Relation to Cell Fusion and?Cancer

Cell fusion, a process that merges two or more cells into one, is required for normal development and has been explored as a tool for stem cell therapy. It has also been proposed that cell fusion causes cancer and contributes to its progression. These functions rely on a poorly understood ability of cell fusion to create new cell types. We suggest that this ability can be understood by considering cells as attractor networks whose basic property is to adopt a set of distinct, stable, self-maintaining states called attractors. According to this view, fusion of two cell types is a collision of two networks that have adopted distinct attractors. To learn how these networks reach a consensus, we model cell fusion computationally. To do so, we simulate patterns of gene activities using a formalism developed to simulate patterns of memory in neural networks. We find that the hybrid networks can assume attractors that are unrelated to parental attractors, implying that cell fusion can create new cell types by nearly instantaneously moving cells between attractors. We also show that hybrid networks are prone to assume spurious attractors, which are emergent and sporadic network states. This finding means that cell fusion can produce abnormal cell types, including cancerous types, by placing cells into normally inaccessible spurious states. Finally, we suggest that the problem of colliding networks has general significance in many processes represented by attractor networks, including biological, social, and political phenomena.     

The Saccharomyces cerevisiae BEM1 homologue in Neurospora crassa promotes co‐ordinated cell behaviour resulting in cell fusion

Directed growth or movement is a common feature of microbial development and propagation. In polar growing filamentous fungi, directed growth requires the interaction of signal sensing machineries with factors controlling polarity and cell tip extension. In Neurospora crassa an unusual mode of cell–cell signalling mediates mutual attraction of germinating spores, which subsequently fuse. During directed growth of the two fusion partners, the cells co‐ordinately alternate between two physiological stages, probably associated with signal sending and receiving. Here, we show that the Saccharomyces cerevisiae BEM1 homologue in N. crassa is essential for the robust and efficient functioning of this MAP kinase‐based signalling system. BEM1 localizes to growing hyphal tips suggesting a conserved function as a polarity component. In the absence of BEM1, activation of MAK‐2, a MAP kinase essential for germling fusion, is strongly reduced and delayed. Germling interactions become highly instable and successful fusion is greatly reduced. In addition, BEM1 is actively recruited around the forming fusion pore, suggesting potential functions after cell–cell contact has been established. By genetically dissecting the contribution of BEM1 to additional various polarization events, we also obtained first hints that BEM1 might function in different protein complexes controlling polarity and growth direction.     

Surface‐directed assembly of cell‐laden microgels

Cell‐laden microscale hydrogels (microgels) can be used as tissue building blocks and assembled to create 3D tissue constructs with well‐defined microarchitecture. In this article, we present a bottom‐up approach to achieve microgel assembly on a patterned surface. Driven by surface tension, the hydrophilic microgels can be assembled into well‐defined shapes on a glass surface patterned with hydrophobic and hydrophilic regions. We found that the cuboidic microgels (～100–200 µm in width) could self‐assemble into defined shapes with high fidelity to the surface patterns. The microgel assembly process was improved by increasing the hydrophilicity of the microgels and reducing the surface tension of the surrounding solution. The assembled microgels were stabilized by a secondary crosslinking step. Assembled microgels containing cells stained with different dyes were fabricated to demonstrate the application of this approach for engineering microscale tissue constructs containing multiple cell types. This bottom‐up approach enables rapid fabrication of cell‐laden microgel assemblies with pre‐defined geometrical and biological features, which is easily scalable and can be potentially used in microscale tissue engineering applications. Biotechnol. Bioeng. 2010; 105: 655–662. © 2009 Wiley Periodicals, Inc.     

Membrane excitability expressed in human neuroblastoma cell hybrids

The voltage-sensitive Na+ channel is responsible for the action potential of membrane electrical excitability in neuronal tissue. Three methods were used to demonstrate the presence of neurotoxin-responsive Na+ channels in two hybrid cell lines resulting from the fusion of excitable human neuroblastoma cells with mouse fibroblasts. Only one of the two electrically active hybrid cell lines maintained the sensitivity of the neuroblastoma parent to tetrodotoxin (TTX). The other hybrid, although electrically active, was not responsive to TTX or scorpion venom. Comparisons of the patterns of expression of membrane excitability and of chromosome complements in these human neuroblastoma cell hybrids suggest that the phenotype of membrane excitability is composed of genetically distinct elements.     

Giant cell tumor of bone: A unique paradigm of stromal–hematopoietic cellular interactions

Giant cell tumor of bone is a progressive, potentially malignant process which destroys skeletal tissue by virtue of its osteoclast complement. As a biological entity it provides a unique natrual model of bone resorption by osteoclasts whose recruitment and development is controlled by a neoplastic population of fibroblast-like cells. Understanding of the etiopathogenesis of this tumor could provide new insights into the mechanisms underlying osteoblast–osteoclast interactions in normal and diseased bone. Recent studies have shown that the stromal cell component in giant cell tumors is the only proliferating subpopulation of cells, and the giant cells themselves are nonproliferative and reactive. These stromal cells express several genes associated with the osteoblastic phenotype, synthesize, to a limited degree, certain matrix proteins associated with bone, and express several factors which are presumably involved in the recruitment of osteoclasts. In culture, giant cell tumor–associated stromal cells promote the fusion of monocytes and the proliferation of osteoblasts either by the secretion of factors or cell–cell contact. Hence, giant cell tumor of bone is a self-contained biosystem in which cells of both the stromal and hematopoietic lineages interact in a fashion similar to that observed in normal skeletal remodeling. The neoplastic nature of the stromal component, however, drives the hematopoietic precursors to undergo fusion, produces aggressive bone resorption, and results in extensive skeletal destruction. Examination of the various components of this system could lead to new directions for investigations aimed at a better understanding of osteoblast–osteoclast interactions. © 1994 Wiley-Liss, Inc.     

Co‐localization of β1,6‐branched Oligosaccharides and Coarse Melanin in Macrophage–Melanoma Fusion Hybrids and Human Melanoma Cells In Vitro

Fusion hybrids between normal macrophages and Cloudman S91 melanoma cells were shown earlier to have increased metastatic potential, along with high expression of β1,6‐N‐acetylglucosaminyltransferase  V and β1,6‐branched oligosaccharides. Curiously, hybrids, but not parental melanoma cells, also produced ‘coarse melanin’– autophagic vesicles with multiple melanosomes. As β1,6‐branched oligosaccharides were known to be associated with metastasis, and coarse melanin had been described in invasive human melanomas, we looked for potential relationships between the two. Using lectin‐ and immunohistochemistry, we analyzed cell lines producing coarse melanin for β1,6‐branched oligosaccharides: gp100/pmel‐17 (a melanosomal structural component) and CD63 (a late endosome/lysosome component associated with melanoma and certain other human cancers). Cell lines used in this study were (i) hybrid 94‐H48, a highly metastatic, macrophage–melanoma experimental fusion hybrid; (ii) 6^neo mouse melanoma cells, the weakly metastatic, parental fusion partner; and (iii) SKmel‐23, a human melanoma cell line derived from a metastasis. Coarse melanin granules were prominent both in hybrids and in SKmel‐23 cells, and co‐localized with stains for β1,6‐branched oligosaccharides, gp100/pmel 17, and CD63. This is the first report of this phenotype being expressed in vitro, although co‐expression of β1,6‐branched oligosaccharides and coarse melanin was recently shown to be a common and pervasive characteristic in archival specimens of human melanomas, and was most prominent in metastases. The results suggest that pathways of melanogenesis in melanoma may differ significantly from those in normal melanocytes. In vitro expression of this phenotype provides new biological systems for more detailed analyses of its genesis and regulation at the molecular genetic level.     

Bone Marrow Transplantation Results in Human Donor Blood Cells Acquiring and Displaying Mouse Recipient Class I MHC and CD45 Antigens on Their Surface

Background

Mouse models of human disease are invaluable for determining the differentiation ability and functional capacity of stem cells. The best example is bone marrow transplants for studies of hematopoietic stem cells. For organ studies, the interpretation of the data can be difficult as transdifferentiation, cell fusion or surface antigen transfer (trogocytosis) can be misinterpreted as differentiation. These events have not been investigated in hematopoietic stem cell transplant models.

Methodology/Principal Findings

In this study we investigated fusion and trogocytosis involving blood cells during bone marrow transplantation using a xenograft model. We report that using a standard SCID repopulating assay almost 100% of the human donor cells appear as hybrid blood cells containing both mouse and human surface antigens.

Conclusion/Significance

Hybrid cells are not the result of cell-cell fusion events but appear to be due to efficient surface antigen transfer, a process referred to as trogocytosis. Antigen transfer appears to be non-random and includes all donor cells regardless of sub-type. We also demonstrate that irradiation preconditioning enhances the frequency of hybrid cells and that trogocytosis is evident in non-blood cells in chimera mice.     

Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential

Most previous studies have linked cancer–macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression.     

Excess cholesterol inhibits glucose‐stimulated fusion pore dynamics in insulin exocytosis

Type 2 diabetes is caused by defects in both insulin sensitivity and insulin secretion. Glucose triggers insulin secretion by causing exocytosis of insulin granules from pancreatic β‐cells. High circulating cholesterol levels and a diminished capacity of serum to remove cholesterol from β‐cells are observed in diabetic individuals. Both of these effects can lead to cholesterol accumulation in β‐cells and contribute to β‐cell dysfunction. However, the molecular mechanisms by which cholesterol accumulation impairs β‐cell function remain largely unknown. Here, we used total internal reflection fluorescence microscopy to address, at the single‐granule level, the role of cholesterol in regulating fusion pore dynamics during insulin exocytosis. We focused particularly on the effects of cholesterol overload, which is relevant to type 2 diabetes. We show that excess cholesterol reduced the number of glucose‐stimulated fusion events, and modulated the proportion of full fusion and kiss‐and‐run fusion events. Analysis of single exocytic events revealed distinct fusion kinetics, with more clustered and compound exocytosis observed in cholesterol‐overloaded β‐cells. We provide evidence for the involvement of the GTPase dynamin, which is regulated in part by cholesterol‐induced phosphatidylinositol 4,5‐bisphosphate enrichment in the plasma membrane, in the switch between full fusion and kiss‐and‐run fusion. Characterization of insulin exocytosis offers insights into the role that elevated cholesterol may play in the development of type 2 diabetes.     

Characteristics of the ultrastructural organization of the nuclei of somatic cell hybrids

A study was made of the ultrastructure of cell nuclei of two types of hybrid clones obtained from the fusion of Chinese hamster with human skin fibroblasts, and from that of mouse hepatoma cells with mink fibroblasts. In cell nuclei of the eight hybrid clones deep invaginations of the inner membrane, not characteristic of the parent cells, were revealed. Analysis of serial sections, and application of electron microscopic radioautography and histochemistry have suggested that these structures are associated with the nuclear envelope which is necessary for regulating the superfluous chromosome localization in the hybrid nucleus.     

Lysolecithin-induced fusion of fibroblasts

Summary Lysolecithin has been used to induce cell fusion between two metabolically deficient mouse fibroblast lines, A₉ and B₈₂. Attempted fusion in suspension led to excessive cell clumping and complete loss of viability. Addition of lysolecithin solutions to confluent monolyers caused extensive detachment of cells from glass or plastic surfaces. At higher levels of lysolecithin few cells survived. When the conditions were controlled (50 to 250 μg per ml for up to 20 min), extensive polykaryocyte formation was observed. In the presence of selective medium (HAT) colonies of hybrid cells grew and a series of cell strains were isolated. The presence of inosinic acid pyrophosphorylase (absent in A₉ cells) was demonstrated in the hybrid cells which were shown to have almost double the cell volume of the parent A₉ and B₈₂ cells. Unlike the parent cell lines, the hybrid cells grew well in the presence of HAT both as monolayers and in suspension.     

草木樨状黄芪和木本霸王的科间体细胞杂交

在成功培养原生质体的基础上,用改进的PEG-高pH高钙法诱导草木樨状黄（Astragalus melilotoides）和木本霸王（Zygophyllum xanthoxylum）原生质体融合,得到了科间体细胞杂种融合细胞。采用罗丹明-6G预处理草木樨状黄芪原生质体以及UV-B辐照霸王原生质体,使双亲原生质体及其同源融合产物均不能持续分裂而死亡,融合后的杂种细胞由于生理互补可恢复持续分裂能力而被筛选出来。融合产物经培养分裂获得了2个杂种细胞系,其中1个分化出芽。染色体计数和分子鉴定证明了杂种的真实性。初步比较了杂种细胞系及亲本对盐分和水分胁迫的耐受性,结果表明杂种细胞系对盐分和水分胁迫的耐受性介于两个亲本之间。     

Activation and Suppression of Melanogenesis in Mouse Melanoma Cells by Hybridization with Chick Embryonic Cells

Sendai virus-induced fusion of 6-thioguanine-resistant mouse melanoma cells (TG14) with various types of chick embryonic tissue cells resulted in the formation of hybrid cells. Isolated hybrid clones possessed almost complete sets of the cell chromosomes of the parent mouse and several dot-like chromosomes of the chick. Each type of hybrid clone showed characteristic tyrosinase activity that resulted in melanin production. An enhanced production of melanin was observed in the hybrids between not highly pigmented TG14 cells and retinal pigment cells. Electrophoretic analyses showed that banding patterns of tyrosinase were not of chick type but of mouse melanoma type. Numerous stage 111 and IV melanosomes of the mouse melanoma type were observed in pigmented hybrid clones. On the other hand, hybrid cells between mouse melanoma cells and chick embryonic liver cells exhibited lower tyrosinase activity.     

Synthetic peptides derived from an N‐terminal domain of the E2 protein of GB virus C in the study of GBV‐C/HIV‐1 co‐infection

Synthetic peptides derived from GB virus C (GBV‐C) have previously been studied in our group for the development of new systems capable of diagnosing diseases caused by this humanotropic virus. We also recently described specific peptide domains of the E2 envelop protein of GBV‐C that have the capacity to interfere with the HIV‐1 fusion peptide, produce a notable decrease in cellular membrane fusion, and perturb HIV‐1 infectivity in a dose‐dependent manner. The present work discloses the design and synthesis of both linear and cyclic branched peptides based on a previously reported N‐terminal sequence of the GBV‐C E2 protein. Immunoassays and cell–cell fusion assays were performed to evaluate their diagnostic value to detect anti‐GBV‐C antibodies in HIV‐1 patients, as well as their putative anti‐HIV‐1 activity as entry inhibitors. Our results showed that chemical modifications of the selected E2(7–26) linear peptide to afford cyclic architecture do not result in an enhanced inhibition of gp41 HIV‐1‐mediated cell–cell fusion nor improved sensitivity in the detection of GBV‐C antibodies in HIV‐1 co‐infected patients. Thus, the ELISA data reinforce the potential utility of linear versions of the E2(7–26) region for the development of new peptide‐based immunosensor devices for the detection of anti‐GBV‐C antibodies in HIV‐1 co‐infected patients. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Efficient GFP‐labeling and analysis of spermatogenic cells using the IRG transgene and flow cytometry

Spermatogenesis is a highly ordered developmental program that produces haploid male germ cells. The study of male germ cell development in the mouse has provided unique perspectives into the molecular mechanisms that control cell development and differentiation in mammals, including tissue‐specific gene regulatory programs. An intrinsic challenge in spermatogenesis research is the heterogeneity of germ and somatic cell types present in the testis. Techniques to separate and isolate distinct mouse spermatogenic cell types have great potential to shed light on molecular mechanisms controlling mammalian cell development, while also providing new insights into cellular events important for human reproductive health. Here, we detail a versatile strategy that combines Cre‐lox technology to fluorescently label germ cells, with flow cytometry to discriminate and isolate germ cells in different stages of development for cellular and molecular analyses.     

Bimolecular fluorescence complementation analysis of eukaryotic fusion products

Background information. Cell fusion is known to underlie key developmental processes in humans and is postulated to contribute to tissue maintenance and even carcinogenesis. The mechanistic details of cell fusion, especially between different cell types, have been difficult to characterize because of the dynamic nature of the process and inadequate means to track fusion products over time. Here we introduce an inducible system for detecting and tracking live cell fusion products in vitro and potentially in vivo. This system is based on BiFC (bimolecular fluorescence complementation) analysis. In this approach, two proteins that can interact with each other are joined to fragments of a fluorescent protein and are expressed in separate cells. The interaction of said proteins after cell fusion produces a fluorescent signal, enabling the identification and tracking of fusion products over time. Results. Long‐term tracking of fused p53‐deficient cells revealed that hybrid cells were capable of proliferation. In some cases, proliferation was preceded by nuclear fusion and division was asymmetric (69%±2% of proliferating hybrids), suggesting chromosomal instability. In addition, asymmetric division following proliferation could give rise to progeny indistinguishable from unfused counterparts. Conclusions. These results support the possibility that the chromosomal instability characteristic of tumour cells may be incurred as a consequence of cell fusion and suggest that the role of cell fusion in carcinogenesis may have been masked to this point for lack of an inducible method to track cell fusion. In sum, the BiFC‐based approach described here allows for comprehensive studies of the mechanism and biological impact of cell fusion in nature.