HUMAN-MOUSE HYBRID CELL LINES CONTAINING PARTIAL COMPLEMENTS OF HUMAN CHROMOSOMES AND FUNCTIONING HUMA N GENES* BY MARY C. WEISSt AND HOWARD GREEN DEPARTMENT OF PATHOLOGY, NEW YORK UNIVERSITY SCHOOL OF MEDICINE Communicated by Boris Ephrussi, June 26, 1967 This paper will describe the isolation and properties of a group of new somatic hybrid cell lines obtained by crossing human diploid fibroblasts with an established mouse fibroblast line. These hybrids represent a combination between species more remote than those previously described (see, however, discussion of virus- induced heterokaryons). They are also the first reported hybrid cell lines con- taining human components and possess properties which may be useful for certain types of genetic investigations. Interspecific hybridizations involving rat-mouse,' hamster-mouse,2 3 and Ar- menian hamster-Syrian hamster4 combinations have been shown to yield popula- tions of hybrid cells capable of indefinite serial propagation. Investigations of the karyotype and phenotype of such hybrids have shown that both parental genomes are present2 5 and functional.6 In every case, some loss of chromosomes has been observed; this occurred primarily during the first few months of propagation, usually amounted to approximately 10-20 per cent of the complement present in newly formed hybrid cells, and involved chromosomes of both parents. Recent studies have provided evidence of preferential loss of chromosomes of one parental species in interspecific hybrids.2'5 A more extreme example of this preferential loss has been encountered in the human-mouse hybrid lines to be described, in which at least 75 per cent, and in some cases more than 95 per cent, of the human complement has been lost. It has been possible to relate the human characteristics of the hybrid phenotype to the number of human chromosomes retained. This has been shown for the colonial morphology of the hybrid and for the human antigens in the hybrid cell membrane, which were detected by mixed cell agglutination. Furthermore, information has been obtained with respect to the chromosomal localization of the thymidine kinase gene. Materials and Methods.-Cell lines: LM (TK-) cl 1-D (hereafter referred to as cl 1-D), a sub- line of mouse L cells, was isolated by Dubbs and Kit7 and provided by Dr. B. Ephrussi. This clone, deficient in thymidine kinase, is resistant to 30 jsg/ml of 5-bromodeoxyuridine (BUDR). WI-38, a diploid strain of human embryonic lung fibroblasts,8 was provided by the American Type Culture Collection and was propagated in monolayer culture in this laboratory for 10 to 20 cell generations before hybridization. Culture method and selection of hybrids: All cultures were maintained in standard growth medium (Dulbecco and Vogt's modification of Eagle's minimal medium containing 10% calf serum), in some cases supplemented with 30 ,ug/ml of BUDR, or hypoxanthine (1 X 10-4 M), aminopterin (4 X 10-7 M), thymidine (1.6 X 10-5 M) (HAT). The selective system used was originally de- scribed by Littlefield9 and has been modified by Davidson and Ephrussi'0 for use with a bio- chemically marked cell line combined with a contact-inhibited strain of diploid fibroblasts. The mouse cell line cl 1-D, deficient in thymidine kinase, is unable to grow in medium containing HAT; no spontaneous reversion to HAT resistance has been observed in this cell line, and it seems most likely that a deletion involving this gene has occurred." The human diploid strain, containing 1104 Downloaded by guest on October 1, 2021 VOL. 58, 1967 GENETICS: WEISS AND GREEN 1105 thymidine kinase activity, is able to grow in this medium but it forms only a relatively thin cell layer, while hybrid cells are able to pile up against this background and form discrete colonies. Karyotype of parental cells: Cl 1-D is characterized by the presence of 51 (50-55) chromosomes, 9 (8-10) of which are large metacentrics. Among the latter is the D chromosome'2 which, owing to the presence of a secondary constriction, appears to be dicentric and has no equivalent in the human complement. As can be seen in Figure 2a, most of the chromosomes of cl 1-D are telo- centric. WI-38 is a diploid human strain;8 the cells contain 46 chromosomes, the pairsl of which may be grouped into seven classes (Denver Classification). Of these, only two (A and G) contain chromosomes which are likely to be confused with the mouse chromosomes (Fig. 2b). Demonstration of cell surface antigens by mixed hemagglutination: These experiments were carried out using a modification of the method described by Kelus, Gurner, and Coombs.13 Immune sera were obtained from rabbits following injection of WI-38 cells in Freund's adjuvant. Com- plement was inactivated at 560 and the serum absorbed twice with a total of 6 X 106 cl 1-D cells per ml of serum. In order to test for the presence of human antigens, 2 X 10' trypsinized paren- tal or hybrid cells were washed in buffered salt solution containing 0.1% bovine serum albumin, incubated with 0.1 ml antiserum for 1 hr at room temperature, again washed three times and mixed with 0.1 ml of a 2% suspension of washed human red blood cells. The mixture was centri- fuged gently, resuspended, and a drop pipetted onto a glass slide for examination. A cover slip placed over the drop was allowed to settle for 5 min, so that erythrocytes attached to the surface of a test cell were forced to its perimeter and appeared as an encircling ring. In controls for specificity of the agglutination, mouse erythrocytes were employed in place of human. Mouse species-specific antigens were identified by the use of rabbit antimouse ascites tumor antiserum and mouse erythrocytes. Results.-Production and identification of hybrid cells: Cultures were initiated with mixtures of 2 X 106 cl 1-D cells and 1 X 104 WI-38 cells. After four days of growth in standard medium, the cultures were placed in selective medium (HAT). The cl 1-D cells degenerated within seven days, leaving a single layer of human cells; after 14 to 21 days, hybrid colonies (Fig. 1) could be detected growing on the human cell monolayer. A number of these were isolated and grown to mass culture. In other cases the entire culture was transferred; within a few weeks the hybrid cells over-grew the remaining human fibroblasts and in the course of serial cultivation all human cells disappeared from the population. Of three independent experiments performed, all yielded hybrid colonies, with a frequency of approximately one per 2 X 104 WI-38 cells. In all hybrids examined 20 generations after their formation, the same karyotypic pattern was found; all (or nearly all) of the expected mouse chromosomes were present, but of the human chromosomes only a minority remained, varying in number from 2 to 15 in the A V m#-F FIG 1. -Phase contrast photomicrographs of living cells of (a) WI-38; (b) cl 1-D; (c) hybrid clone HM-2 P2. The parallel orientation of the highly elongated WI-38 cells is in contrast to the random orientation of the highly refractile and less fusiform cells of cl 1-D. The cells of the hybrid clone, while highly refractile, have a degree of orientation intermediate between cl 1-D and WI-38. Downloaded by guest on October 1, 2021 1106 GENETICS: WEISS AND GREEN PROC. N. A. S. various populations. The mean numbers of human chromosomes found in six independent hybrid clones were as follows: 6.5, 2.0, 11.6, 10.9, 3.0, and 3.1. An occasional hybrid mitosis contained more than one copy of the same human chromo- some, but most contained only one member of a given pair. The karyotype of a hybrid cell of clone HM\1-2 is shown in Figure 2c. Loss of human thymidine kinase gene(s) from hybrid cells: Since survival of cells in HAT medium requires the presence of thymidine kinase, all hybrid cells selected in these experiments presumably contain the human gene(s) for this enzyme. This conclusion is supported by the fact that the cells can be killed by growth in the presence of BUDR. During continued propagation in HAT, any variants which may have lost this gene are eliminated. However, such variants occur with high frequency and were obtained selectively in a single step by transfer of the population to a medium containing BUDR, which eliminated most of the popula- tion and permitted the growth only of cells without thymidine kinase activity. As expected, these BUDR-resistant variant hybrid cells were not able to grow in HAT medium. They did retain many, but not all, of the human chromosomes which were present before the selection (see below). Cells resistant to BUDR also appeared in cultures propagated in standard medium without HAT and seemed to enjoy some selective advantage, for after several months of serial prop- agation they amounted to about 50 per cent of the population, as measured by HAT sensitivity. As no revertants to HAT resistance were obtained, BUDR resist- ance probably occurred by deletion. XX fly O* 7X6 FIG. 2.-Chromosomes of \ C1 l s Z z | 1-5A 14-5) 1-D, WI -38, and hybrids. (a) :i^:iI: Karyotype mouse parental line,el 1-D. Nine long meta- i6-2i centric chromosomes are present, I i A AaaI I I g one of which (arrow, the D X X * * chromosome) is identifiable in 0uI m o sm A A et a A ^ AA IL all chromosome preparations. ^^ ***X CI13-iS E1618 The karyotype also shows 43 8B~A~i~gOI*telocentric chromosomes, none l a .A of which contains distinguishable F!19-20 G i2l-22 satellites.