Cell Fusion Products Research Articles

In Vivo Cell Fusion between Mesenchymal Stroma/Stem-Like Cells and Breast Cancer Cells.

Cellular communication within the tumor microenvironment enables important interactions between cancer cells and recruited adjacent populations including mesenchymal stroma/stem-like cells (MSC). These interactions were monitored in vivo following co-injection of GFP-labeled human MSC together with mcherry-labeled MDA-MB-231 breast cancer cells in NODscid mice. Within 14 days of tumor development the number of initially co-injected MSC had significantly declined and spontaneous formation of breast cancer/MSC hybrid cells was observed by the appearance of double fluorescing cells. This in vivo fusion displayed a rare event and occurred in less than 0.5% of the tumor cell population. Similar findings were observed in a parallel in vitro co-culture. Characterization of the new cell fusion products obtained after two consecutive flow cytometry cell sorting and single cell cloning revealed two populations, termed MDA-hyb3 and MDA-hyb4. The breast cancer fusion cells expressed both, GFP and mcherry and displayed more characteristics of the MDA-MB-231 cells than of the parental MSC. While little if any differences were determined in the proliferative capacity, a significant delay of MDA-hyb3 cells in tumor formation was observed when compared to the parental MDA-MB-231 cells. Moreover, MDA-hyb3 cells developed an altered pattern of distant organ metastases. These findings demonstrated dynamic tumor changes by in vivo and in vitro fusion with the development of new breast cancer hybrid cells carrying altered tumorigenic properties. Consequently, cancer cell fusion contributes to progressively increasing tumor heterogeneity which complicates a therapeutic regimen.

Remembrances of Hilary Koprowski from a Surgeon's Perspective.

Remembrances of Hilary Koprowski from a Surgeon's Perspective.

Cell fusion in tumor progression: the isolation of cell fusion products by physical methods.

BackgroundCell fusion induced by polyethylene glycol (PEG) is an efficient but poorly controlled procedure for obtaining somatic cell hybrids used in gene mapping, monoclonal antibody production, and tumour immunotherapy. Genetic selection techniques and fluorescent cell sorting are usually employed to isolate cell fusion products, but both procedures have several drawbacks.ResultsHere we describe a simple improvement in PEG-mediated cell fusion that was obtained by modifying the standard single-step procedure. We found that the use of two PEG undertreatments obtains a better yield of cell fusion products than the standard method, and most of these products are bi- or trinucleated polykaryocytes. Fusion rate was quantified using fluorescent cell staining microscopy. We used this improved cell fusion and cell isolation method to compare giant cells obtained in vitro and giant cells obtained in vivo from patients with Hodgkin's disease and erythroleukemia.ConclusionsIn the present study we show how to improve PEG-mediated cell fusion and that cell separation by velocity sedimentation offers a simple alternative for the efficient purification of cell fusion products and to investigate giant cell formation in tumor development.

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.

Human progenitor cells with high aldehyde dehydrogenase activity efficiently engraft into damaged liver in a novel model†

Human cord blood stem cells (hCBSCs) have been reported to generate hepatocyte-like cells and thus hold promise for repairing damaged liver. However, the frequency of hCBSC-derived hepatocytes varies tremendously between different studies, and it is still controversial as to whether hCBSC-derived cells can transdifferentiate into hepatocytes or simply fuse to recipient hepatocytes. We used the beta-glucuronidase-deficient nonobese diabetic/severe combined immunodeficient/mucopolysaccharidosis type VII (NOD/SCID/MPSVII) mouse model for better identification of engrafted cells. We transplanted lineage-depleted human umbilical cord blood-derived cells with high aldehyde dehydrogenase activity (ALDH(hi)Lin(-)) into irradiated NOD/SCID/MPSVII mice followed by carbon tetrachloride administration to induced liver damage. ALDH(hi)Lin(-) cells were efficiently engrafted in the recipient mouse livers and improved recovery of the mice from toxic insult. The percentage of human cells in these livers ranged between 3% and 14.2% using quantitative real-time polymerase chain reaction. Furthermore, human-originated cells expressing liver-specific alpha1-antitrypsin messenger RNA, albumin and hepatocyte nuclear factor 1 protein were detected in the recipient livers. Interestingly, human versus murine centromeric fluorescent in situ hybridization analysis on the liver sections demonstrated that most human cells were not fused to mouse cells. However, the majority of the human originated albumin-expressing cells also carried mouse genetic material, hence were the product of cell fusion. hCBSCs or their progeny may home to the injured liver and release trophic factors that hasten tissue repair, whereas fusion of these cells with hepatocytes may occur rarely and contribute to a lesser extent to liver repair.

Donor Origin of Multipotent Adult Progenitor Cells in Radiation Chimeras.

Multipotent Adult Progenitor Cells (MAPC) are bone marrow derived stem cells that can be extensively expanded in vitro and can differentiate in vivo and in vitro into cells of all three germinal layers: ectoderm, mesoderm, endoderm. The origin of MAPC within bone marrow (BM) is unknown. MAPC are believed to be derived from the BM stroma compartment as they are isolated within the adherent cell component. Numerous studies of bone marrow chimeras in human and mouse point to a host origin of bone marrow stromal cells, including mesenchymal stem cells. We report here that following syngeneic bone marrow transplants into lethally irradiated C57Bl/6 mice, MAPC are of donor origin. When MAPC were isolated from BM chimeras (n=12, 4–12 weeks post-syngeneic BM transplant from a transgenic mouse ubiquitously expressing GFP), a mixture of large and small GFP-positive and GFP-negative cells were seen early in culture. While the large cells stained positive for stroma cell markers (smooth muscle actin), mesenchymal stem cell makers (CD73, CD105, CD44) or macrophages (CD45, CD14), the small cells were negative for all these markers and after 30 cell doublings, these cells displayed the classical phenotype of MAPC (CD45−,CD105−, CD44−, CD73−, FLK-1+(vascular endothelial growth factor receptor 2, VEGFR2), Sca-1+,CD13+).In a second experiment, BM obtained one month post BM transplant (n=3) was harvested and mononuclear cells were sorted as GFP-positive and GFP-negative cells and were cultured in MAPC expansion medium. MAPC grew from the GFP-positive fraction. These GFP positive cells displayed the typical MAPC-like immunophenotypes, displayed a normal diploid karyotype and were expanded for more than 50 cell doublings and differentiated into endothelial cells, hepatocytes and neurons. To rule out the possibility that MAPC are the product of cell fusion between a host and a donor cell either in vivo or in our in vitro culture conditions, we performed sex mismatched transplants of female GFP donor BM cells into a male host. BM from 5 chimeras were harvested 4 weeks after transplant and MAPC cultures were established. MAPC colonies were then sorted as GFP-positive and GFP- negative and analyzed for the presence of Y-chromosome by FISH analysis. As expected all GFP-negative (host cells) contained the Y-chromosome whereas all GFP-positive cells (donor cells) were negative for the Y-chromosome by FISH. This proves that MAPC are not derived from an in vitro or in vivo fusion event.In a third study, BM mononuclear cells from mice that had been previously BM-transplanted with syngeneic GFP-positive donors (n=3) were transplanted into a second set of syngeneic recipients (n=9). Two months after the second transplant, BM was harvested and mononuclear cells were cultured in MAPC medium. The secondary recipients also contained GFP-positive MAPC. This is the first demonstration that BM transplantation leads to the transfer of cells that upon isolation in vitro generate MAPCs and, whatever the identity of this cell may be, is eliminated by irradiation. We believe this is an important observation as MAPC hold great clinical potential for stem cell and/or gene therapy and, thus, BM transplant may serve as a way to deliver and reconstitute the MAPC population. In addition, this study provides insight into the nature of MAPC. The capacity to be transplantable within unfractionated BM transplant renders a functional and physiological distinction between MAPC and BM stromal cells. This study validates the use of unfractionated BM transplants to study the nature and possible in vivo role of MAPC in the BM.

Getting Melanoma Cells to Stimulate With Frequency

Strategies for the delivery of antigenic peptides to patients with advanced malignant melanoma include a variety of novel approaches, such as the administration of recombinant DNA-encoding antigenic-peptide sequences; mRNA transcripts and peptides collected directly from patient tumors; recombinant replication-deficient adenoviral and retroviral vectors encoding tumor antigens; synthetic peptides modified for enhanced binding to antigenpresenting cell (APC) and human leukocyte antigen (HLA) molecules; allogeneic tumor cell lysates; peptide-loaded dendritic cells; tumor cell-dendritic cell fusion products; and irradiated autologous tumor cell preparations. Thus far, it is not clear that any one method is superior for the generation of antigen-specific T cells, and the induction of durable clinical responses has remained an elusive goal. It may develop that multiple methods of vaccination will be employed depending upon the specific disease setting or the type of antigen that is being delivered. One area of research thatmayenhance the successof vaccinationstrategies is the development of improved vaccine adjuvants. The development of an effective immune response and long-lasting immunologic memory in response to the delivery of an antigenic peptide requires the generation of a pool of antigen-specific T cells. Coadministration of an adjuvant with the vaccine can enhance the activity of weak immunogens. Vaccine adjuvants may function in several ways. An adjuvant may act as a depot for the vaccine, thus prolonging the time period over which an antigen is presented to APCs. Alternatively, an adjuvant may be employed as a means to change the character, number, or activation state of APCs at the vaccination site. Finally, an adjuvant may be used to alter the immunologic pathway by which the protein is being presented or processed, thus steering the immune system toward either a cellbased or an antibody-based response to the antigen. Cytokines are of particular interest to this field because they affect which arm of the immune system is stimulated to respond to the antigenic stimulus. Interleukin-12, interleukin-2 (IL-2), and interferonare examples of cytokines that have been tested as adjuvants in the setting of metastatic malignant melanoma. However, laboratory and clinical studies indicate that granulocyte-macrophage colony-stimulating factor (GM-CSF) may have particular advantages as a vaccine adjuvant. GM-CSF stimulates the activation, maturation, and migration of dendritic cells (DCs) and macrophages and induces their expression of class II major histocompatibility complex molecules. Using a murine model, Disis et al showed that GM-CSF was comparable to complete Freund’s adjuvant in augmenting the immune response to both tetanus toxoid and a peptide sequence derived from the rat neu protein (homologue of human c-erb-B2). Importantly, peptides administered without GM-CSF were essentially nonimmunogenic. Dranoff et al used a murine melanoma model to evaluate seven different cytokines for their ability to enhance the immunogenicity of irradiated B16 tumor cells, which alone do not stimulate significant antitumor immunity. Tumor cells transduced with a retroviral construct for GM-CSF exerted potent, long-lasting, and specific antitumor immunity that required both CD4 and CD8 T cells. Tumor cells expressing other cytokines were either poorly immunogenic (IL-2, IL-5, interferon[IFN], tumor necrosis factor[TNF]), or weakly immunogenic (IL-4 and IL-6). Vaccination of stage IV melanoma patients with irradiated, autologous melanoma cells engineered to secrete GM-CSF by adenovirus gene transfer led to an antigen-specific T-cell response (as measured by a delayed-type hypersensitivity reaction to nontransduced tumor cells) and measurable lymphocytic infiltration of tumor deposits in a significant proportion of patients, as well as one complete response. Likewise, a multipeptide melanoma vaccine elicited superior T-cell immunity when injected as an emulsion with GM-CSF as compared VOLUME 23 d NUMBER 35 d DECEMBER 1

Stable reprogrammed heterokaryons form spontaneously in Purkinje neurons after bone marrow transplant.

Heterokaryons are the product of cell fusion without subsequent nuclear or chromosome loss. Decades of research using Sendai-virus or polyethylene glycol (PEG)-mediated fusion in tissue culture showed that the terminally differentiated state of a cell could be altered. But whether stable non-dividing heterokaryons could occur in animals has remained unclear. Here, we show that green fluorescent protein (GFP)-positive bone-marrow-derived cells (BMDCs) contribute to adult mouse Purkinje neurons through cell fusion. The formation of heterokaryons increases in a linear manner over 1.5 years and seems to be stable. The dominant Purkinje neurons caused the BMDC nuclei within the resulting heterokaryons to enlarge, exhibit dispersed chromatin and activate a Purkinje neuron-specific transgene, L7-GFP. The observed reprogrammed heterokaryons that form in brain may provide insights into gene regulation associated with cell-fate plasticity.

T-DNA-tagged chromosome 12 in donor Lycopersicon esculentum × L. pennellii is retained in asymmetric somatic hybrids with recipient Solanum lycopersicoides

Asymmetric somatic hybrid plants were recovered after fusing irradiated mesophyll protoplasts of donor Lycopersicon esculentum × L. pennellii (EP) interspecific hybrid with callus-derived protoplasts of recipient Solanum lycopersicoides. EP plant A54 had been previously transformed by an agrobacterium vector, and the T-DNA insert mapped to the L. esculentum chromosome 12. The T-DNA insert conferred kanamycin resistance to EP that was subsequently used to select cell fusion products and recover asymmetric hybrid plants that retained tagged chromosome 12. Doses of 50- and 100-Gy irradiation promoted the elimination of only a few donor chromosomes. At 200 Gy, the regenerated plants had ploidy levels higher than tetraploid. However, the T-DNA tagged chromosome 12 was always retained in the asymmetric hybrid plants tested. Likewise, all plants from the 100-Gy series, with the exception of number 160, were mixoploid in the root-tip cells. Such mixoploid asymmetric somatic hybrids could be stabilized by inducing adventitious shoots on leaf strips cultured on shoot regeneration medium containing kanamycin. The asymmetric hybrid plants did not produce viable seed when self-pollinated or backcrossed to tomato or S. lycopersicoides.

ISOZYME VARIATION AND GENETICS IN ASPARAGUS OFFICINALIS

We have used isozyme techniques (SGE) to assess variation and begin construction of a genetic map of the Asparagus officinalis genome. Isozyme extraction buffers, electrophoretic buffer systems, and isozyme stability during storage were evaluated. Isozyme expression under different environmental conditions was also examined. Thirty-four enzymes were evaluated for their usefulness as genetic markers in A. officinalis. Of these 34, 13 had sufficient activity and resolution on the gels for isozyme analysis. Of the 13 enzyme systems resolved, polymorphisms were observed in aconitase, endopeptidase, malate dehydrogenase, phosphoglucomutase, and shikimate dehydrogenase. Segregation of putative alleles is presented for ACON, END, MDH, PGM and SKDH isozymes. Co-segregation data showed linkage between a SKDH locus and a PGM locus. The isozyme analysis also included Asparagus densiflorus `Sprengeri' and revealed that aspartate aminotransaminase, endopeptidase, and triosephosphate isomerase would be potentially useful for verification of cell fusion products between the two species.

18] Toxin — antitoxin selection for isolating heterokaryons and cell hybrids

Publisher Summary This chapter discusses the methods used in toxin–antitoxin selection for isolating heterokaryons and cell hybrids. The behaviors of cell hybrids have employed genetic selection techniques, such as the HAT system, to isolate cell fusion products. Some “half-selective” strategies are devised where an established immortal cell line is fused to a normal diploid cell type which does not divide in culture, in which it is unnecessary to select against the non dividing normal diploid parental cell type. However, the inability to use genetic selection techniques with normal cells frequently represents a major limitation for many types of studies. Two alternative systems for isolating heterokaryons and cell hybrids developed are discussed in the chapter. The first one exploits the different specificities of two irreversible biochemical inhibitors. Iodoacetamide is directed primarily toward sulfhydryl groups, while diethyl pyrocarbonate reacts mainly with histidine residues. An elegant technique for introducing macromolecules into cells is introduced. Cells are first exposed to a hypenonic sucrose solution containing the molecule. Normally, the pinocytic vesicles that are formed during this period would be transported to the lysosomes, where their contents would be degraded.

Toxin-antitoxin selection for isolating somatic cell fusion products between any cell types.

A selective system was developed that permits the isolation of heterokaryons and cell hybrids between any cell types without the use of selectable genetic markers. Antibodies were introduced into two populations of non-transformed normal diploid human fibroblasts by the osmotic lysis of pinosomes containing hypertonic antibody solutions. Heterokaryons formed by fusing antiricin-injected cells to antidiphtheria toxin-injected cells survived an overnight treatment in both ricin and diphtheria toxin. This approach should have general applicability to a variety of investigations such as the complementation analysis of human genetic syndromes, heterokaryon studies of cell differentiation, tumorigenesis and growth control, cell reconstruction experiments, and the production of hybridoma cells.

RARE-CELL FUSION EVENTS BETWEEN DIPLOID AND HAPLOID STRAINS OF THE SEXUALLY AGGLUTINATIVE YEAST HANSENULA WINGEI

Diploids of the yeast Hansenula wingei are nonagglutinative and do not form zygotes in mixed cultures with either sexually agglutinative haploid mating type. However, a low frequency of diploid x haploid cell fusions (about 10(-3)) is detectable by prototrophic selection. This frequency of rare diploid x haploid matings is not increased after the diploid culture is induced for sexual agglutination. Therefore, we conclude that genes that repress mating are different from those that repress sexual agglutination.--Six prototrophs isolated from one diploid x haploid cross had an average DNA value (microg DNA per 10(8) cells) of 6.19, compared to 2.53 and 4.35 for the haploid and diploid strains, respectively. Four prototrophs were clearly cell-fusion products because they contained genes from both the diploid and the haploid partners. However, genetic analysis of the prototrophs yielded results inconsistent with triploid meiosis; all six isolates yielded a 2:2 segregation for the mating-type alleles and linked genes.--Mitotic segregation of monosomic (2n-1) cells lacking one homolog of the chromosome carrying the mating-type locus is proposed to explain the rare production of sexually active cells in the diploid cultures. Fusion between such monosomic cells and normal haploids is thought to have produced 3n-1 cells, disomic for the chromosome carrying the mating-type locus. We conclude that in the diploid strain we studied, the physiological mechanisms repressing sexual agglutination and conjugation function efficiently, but events occuring during mitosis lead to a low frequency of genetically altered cells in the population.

Increased polyethylene glycol-mediated fusion competence in mitotic cells of a mouse lymphoid cell line

Spontaneous mitotic cells of the mouse leukemic cell line GF7 are preferentially included in cell fusion products after treatment with polyethylene glycol (PEG). This implies a unique configuration of the natural mitotic membrane which is particularly vulnerable to induction of fusion by PEG. Colcemid-arrested GF7 mitotic cells, however, are excluded from PEG-induced cell fusion products, suggesting that colcemid reverses the membrane configuration which is susceptible to the action of PEG. When Sendai virus is used as the fusogenic agent, both colcemid-arrested and spontaneous mitotic cells are selectively fused. There must, therefore, be an essential membrane-fusogen reaction which is characteristically different for each of these agents.

Heterokaryons in the analysis of genes and gene regulation

Cytological and chemical analysis of heterokaryons, the immediate product of cell fusion, offer new possibilities for studying the factors responsible for genetic regulation in eukaryotic cells. In comparison with proliferating cell hybrids the heterokaryon state offers the important advantage that a heterokaryon contains two complete genomes since chromosome loss does not occur, but since segregation and recombination are absent, heterokaryons cannot be used for gene mapping in the same way as proliferating cell hybrids. However, if two cell types carrying different genetic defects are fused the analysis can be used for studies of gene complementation. The biological information obtained with heterokaryons has emphasized the role of the cytoplasm in the control of nuclear activity. When a G1 nucleus is brought into contact with the cytoplasm of an S phase cell the G1 nucleus is stimulated to synthesize DNA. If the nucleus is brought into a mitotic cell, the chromatin of the G1 nucleus is forced to condense into prematurely condensed chromosomes. Inactive nuclei such as the dormant chick erythrocyte nucleus will be stimulated to initiate RNA and DNA synthesis when brought into contact with an active cytoplasm by cell fusion. Specific nuclear proteins have been shown to be responsible for this process of reactivation. Other inactive nuclei such as the nuclei of macrophages and spermatozoa have likewise been shown to be reactivated by fusion with active cells. The degree of activation in all of these cases appears to be determined by the state of the active cell. Inactive nuclei are activated to the same level as the active nucleus but seldom beyond this level. If differentiated cells are fused with undifferentiated cells, usually the differentiated character is lost rapidly after fusion. This observation is in agreement with several studies on proliferating cell hybrids indicating some type of negative control of differentiated properties. In heterokaryons obtained by fusion of cells of a similar type of histotypic differentiation usually coexpression of the differentiated markers is observed.

Autoradiographic Evidence for the Occurrence of Cell Fusion in Cultures of Theileria-Infected Bovine Lymphoid Cells

Theileria parva-infected bovine lymphoid cells, grown in tissue culture, were labelled with tritiated thymidine. Aliquots of this culture were added to equal volumes of unlabelled cultures of T. parva and T. lawrencei. Smears were prepared from the pure and mixed cultures and examined by autoradiography. Virtually all the cells in the pure labelled cultures had taken up the tritiated thymidine, and in many cells theilerial macroschizonts were also labelled. Some of the multinucleate cells in mixed cultures appeared to be the product of cell fusion since they contained both labelled and unlabelled nuclei.

DNA synthesis and myogenesis

Differentiating muscle cells synthesizing myosin, the meromyosins, and actin do not concurrently synthesize DNA. Presumptive myoblasts which synthesize DNA do not concurrently synthesize myosin, the meromyosins or actin. The multinucleated skeletal muscle fiber is the product of cell fusion.