THE manufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest. There are at present a considerable number of permanent cultures of myeloma cells1,2 and screening procedures have been used to reveal antibody activity in some of them. This, however, is not a satisfactory source of monoclonal antibodies of predefined specificity. We describe here the derivation of a number of tissue culture cell lines which secrete anti-sheep red blood cell (SRBC) antibodies. The cell lines are made by fusion of a mouse myeloma and mouse spleen cells from an immunised donor. To understand the expression and interactions of the Ig chains from the parental lines, fusion experiments between two known mouse myeloma lines were carried out.

Continuous cultures of fused cells secreting antibody of predefined specificity

Plasticity of Adult Stem Cells

Cells from established tissue culture lines of mouse ( cIID ) and human ( VA-2 ) origin were fused together with Sendai virus, producing heterokaryons bearing both mouse and human surface antigens which were then followed by the indirect fluorescent antibody method. Within 40 mm following fusion, total mixing of both parental antigens occurred in over 90% of the heterokaryons. Mouse H-2 (histocompatibility) and human surface antigens were visualized by successive treatment of the heterokaryons with a mixture of mouse alloantiserum and rabbit anti- VA-2 antiserum, followed by a mixture of fluorescein-labelled goat anti-mouse IgG and tetramethyl-rhodamine-labelled goat anti-rabbit IgG(Fc). The cIID x VA-2 fusions were carried Out in suspension and maintained at 37°C in a shaking water bath; aliquots were removed at various intervals and stained with the above reagents. The heterokaryon population was observed to change from an initial one (5-min post-fusion) of non-mosaics (unmixed cell surfaces of red and green fluorescence) to one of over 90% mosaics (total intermixing of the 2 fluorochromes) by 40 min after fusion. Mouse-human hybrid lines, derived from similar fusions, gave fluorescence patterns identical to those of the mosaic heterokaryons. Four possible mechanisms would yield such results: (i) a very rapid metabolic turnover of the antigens; (ii) integration of units into the membrane from a cytoplasmic precursor pool; (iii) movement, or ‘diffusion’of antigen in the plane of the membrane; or (iv) movement of existing antigen from one membrane site into the cytoplasm and its emergence at a new position on the membrane. In an effort to distinguish among these possibilities, the following inhibitor treatments were carried out: (1) both short- and long-term (6-h pre-treatment) inhibition of protein synthesis by puromycin, cycloheximide, and chloramphenicol; (2) short-term inhibition of ATP formation by dinitrophenol (DNP) and NaF; (3) short- and long-term inhibition of glutamine dependent pathways with the glutamine analogue 6-diazo-5-oxonorleucine; and (4) general metabolic suppression by lowered temperature. The only treatment found effective in preventing the mosaicism was lowered temperature, from which resulted a sigmoidal curve for per cent mosaics versus incubation temperature. These results would be consistent with mechanisms iii and/or iv but appear to rule out i and ii. From the speed with which the antigen markers can be seen to propagate across the cell membrane, and from the fact that the treatment of parent cells with a variety of metabolic inhibitors does not inhibit antigen spreading, it appears that the cell surface of heterokaryons is not a rigid structure, but is ‘fluid’ enough to allow free ‘diffusion’ of surface antigens resulting in their intermingling within minutes after the initiation of fusion.

The rapid intermixing of cell surface antigens after formation of mouse-human heterokaryons.

The stable states of differentiated cells are now known to be controlled by dynamic mechanisms that can easily be perturbed. An adult cell can therefore be reprogrammed, altering its pattern of gene expression, and hence its fate, to that typical of another cell type. This has been shown by three distinct experimental approaches to nuclear reprogramming: nuclear transfer, cell fusion and transcription-factor transduction. Using these approaches, nuclei from 'terminally differentiated' somatic cells can be induced to express genes that are typical of embryonic stem cells, which can differentiate to form all of the cell types in the body. This remarkable discovery of cellular plasticity has important medical applications.

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Nuclear reprogramming to a pluripotent state by three approaches

DNA synthesis and mitosis are inducible in multinucleate HeLa cells formed by fusion between cells in different phases of the life cycle.

Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis.

Hybrid Cells Derived from Mouse and Man : Artificial Heterokaryons of Mammalian Cells from Different Species

Hybrid cells derived from mouse and man: artificial heterokaryons of mammalian cells from different species.

A virus, inactivated by ultraviolet light, was used to fuse together cells from different species of vertebrate, and the resulting heterokaryons were examined by autoradiographic and cytological techniques. Heterokaryons could be made with both differentiated and undifferentiated cells: HeLa and Ehrlich ascites cells were studied as examples of undifferentiated cells; rabbit macrophages, rat lymphocytes and hen erythrocytes as examples of differentiated cells. These last three cells were chosen because in them, in varying degrees, the process of differentiation has resulted in suppression of the synthesis of DNA or of both DNA and RNA. This suppression was in all cases found to be reversible: the dormant nuclei could be induced to resume the synthesis of RNA or DNA or both when the differentiated cells were fused with a cell which normally synthesizes RNA and DNA. Observations on heterokaryons in which differentiated cells were fused with HeLa cells and with each other permitted certain general conclusions to be drawn about the regulation of nucleic acid synthesis in the heterokaryon. It was found that if either one of the parent cells normally synthesized RNA, RNA synthesis took place in both types of nuclei in the heterokaryon. If either of the parent cells normally synthesized DNA, DNA synthesis took place in both types of nuclei in the heterokaryon. If neither of the parent cells synthesized DNA, no DNA synthesis took place in the heterokaryon. In all cases where a cell which synthesized a particular nucleic acid was fused with one which did not, the active cell initiated the synthesis of this nucleic acid in the inactive partner. In no case did the inactive cell suppress synthesis in the active partner. The nuclei of heterokaryons in which DNA synthesis took place underwent mitosis, and those nuclei which entered mitosis synchronously usually fused together. This process resulted in the progressive formation of mononucleate hybrid cells, which might thus contain within a single nucleus chromosomal complements derived from different species. These mononucleate hybrid cells were also capable of RNA and DNA synthesis, and many of them in turn underwent mitosis. At metaphase these cells showed, in various combinations, the chromosomal complements of the two parent cells. Mononucleate hybrid cells formed by the fusion of a large number of single cells did not appear to be capable of continued multiplication; but mononucleate cells containing one chromosomal set from each parent cell were still found to be undergoing mitosis many days after cell fusion.

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Artificial heterokaryons of animal cells from different species.

POSITIVE results were obtained in experiments designed to show mixed agglutination1 between HeLa cells infected with herpes simplex virus, then treated with rabbit antiherpes serum, and sheep erythrocytes sensitized with rabbit anti-sheep erythrocyte serum and sheep anti-rabbit-γ-globulin serum. The sheep erythrocytes were sensitized by a method fundamentally similar to that of Milgrom et al.2. These results were in contrast to the negative results reported by Fagraeus and Espmark3 in mixed haemadsorption experiments with cells infected with herpes simplex virus.

Adsorption of sensitized sheep erythrocytes to hela cells infected with herpes simplex virus.

THE use of a hybrid between Ehrlich ascites tumour and hamster cell lines to immunize mice against the tumour has been reported1. We report here the use for the same purpose of a hybrid line between Ehrlich ascites tumour cells and cells derived from mouse embryo cells obtained from an inbred strain of mice (CBA). This hybrid is less immunologically foreign than the hamster × Ehrlich hybrid for the randomly bred Swiss mice used in the immunization experiments; it differs antigenically only in histocompatibility antigens, in particular in its H2 antigen, which, for CBA mice, is the H2k antigen. Such a hybrid gave the opportunity of investigating whether the immunity arising after injection of Ehrlich × hamster hybrids1 was the result of strong histocompatibility antigens (of H2 type) in the Ehrlich cells.

Evidence against the Presence of H2 Histocompatibility Antigens in Ehrlich Ascites Tumour Cells

A hybrid of Ehrlich ascites tumour cells and polyoma-transformed hamster cells can be used to immunize mice against the Ehrlich ascites tumour. The hybrid cell is analogous to an attenuated tumour cell and this method of tumour immunization can be adapted for the detection of tumour antigens.

J. F. Watkins

Papers

Hybrid cells derived from mouse and man: artificial heterokaryons of mammalian cells from different species.

Artificial heterokaryons of animal cells from different species.

Adsorption of sensitized sheep erythrocytes to hela cells infected with herpes simplex virus.

Evidence against the Presence of H2 Histocompatibility Antigens in Ehrlich Ascites Tumour Cells

Immunization of Mice against Ehrlich Ascites Tumour using a Hamster/Ehrlich Ascites Tumour Hybrid Cell Line