Differentiation In Vitro of Human-Mouse Teratocarcinoma Hybrids

Vol. 3, No. 12

MOLECULAR AND CELLULAR BIOLOGY, Dec. 1983, p. 2259-2270

0270-7306/83/122259-12$02.00/0 Copyright C 1983, American Society for Microbiology

Differentiation In Vitro of Human-Mouse Teratocarcinoma Hybrids F. J. BENHAM,* M. V. WILES, AND P. N. GOODFELLOW

Human Molecular Genetics Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, England Received 18 April 1983/Accepted 31 August 1983

The mouse embryonal carcinoma (EC) line, PCC4, was used to construct a series of somatic cell hybrids which contain a single or a few human chromosomes. The hybrids all retained the EC phenotype as determined by morphology, expression of SSEA-1, lack of cell surface H-2 antigen and cytokeratin filaments, high alkaline phosphatase levels, the ability to form EC tumors ectopically in nude mice, and the ability to differentiate in response to retinoic acid. Constitutively differentiated cloned lines were derived from retinoic acid-treated hybrid cultures. Several derived lines had a phenotype indistinguishable from that of parietal endoderm cells, which includes synthesis of large amounts of laminin, type IV procollagen, and plasminogen activator. One differentiated line showed a fibroblast-like morphology. The differentiated lines derived from two of the hybrids, MCP6 and GEOC4, stably maintained the sole human chromosomal component present in the EC progenitors. These EC hybrids therefore provide a system to study developmental regulation of the introduced and stably maintained human genetic material derived from a variety of cell types.

Little is known about mechanisms by which the pluripotent embryonic genome promotes expression of new tissue-specific functions as differentiation progresses or how developmental potential becomes restricted to the expression of functions appropriate only to specialized cell lineages. In humans, the study of gene activity in embryonic developmnent is limited to histological description and to the investigation of cultured teratocarcinoma-derived cell lines. These human teratocarcinoma lines do not, however, show extensive differentiation either in vitro or when grown as tumors in nude mice (2). In contrast to the study of human embryogenesis, knowledge of murine embryogenesis is more advanced because of the accessibility to mouse embryos and the availability of the teratocarcinoma-derived cell lines which differentiate well in vitro and in vivo (42-45). The stem cells of teratocarcinomas, called embryonal carcinoma (EC) cells, can be grown as pure cultures in vitro (20, 42). These lines have been used extensively as models for early murine development since they share antigenic, biochemical, and morphological characteristics with early undifferentiated embryonic cells (20, 43), and there are several pluripotent EC lines which will undergo differentiation into derivatives of all three germ layers in vitro and in vivo at ectopic sites (16, 23, 43, 44). Their validity as a model system has been substantiated by the ability of some EC lines to

participate in the development of normal chimeric mice after injection into blastocysts (11, 49, 52). One approach to investigate ways in which the murine embryonic genome regulates gene expression has been to construct somatic cell hybrids between murine EC cells and various nonmurine somatic cells (13, 27, 28). The developmental capacity of such interspecific hybrid cells has been assessed by using them in blastocyst injection to form chimeric mice. Hybrids which contained several chromosomes from a rat hepatoma did contribute to some fetal and adult tissues, and the detection of several ratspecific gene products indicated that modulation of the xenogeneic genes had occurred during organogenesis (13, 27). In a different series of experiments, hybrids which contained a single human chromosome were also shown to participate partially in the development of adult mice; however, unequivocal evidence for retention and participation of the human genetic material was not obtained (28). We have used microcell and conventional fusion techniques to introduce into a mouse EC line, PCC4, a single or a few human chromosomes. In this report, we demonstrate that the three hybrid cell lines retained an EC phenotype. The hybrid lines underwent differentiation in vitro into a variety of cell types in a manner similar or identical to that of the parent mouse EC line, and constitutively differentiated

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VOL. 3, 1983

DIFFERENTIATION OF TERATOCARCINOMA

lines which retained the selected human chromosome present in two EC cell progenitors have been derived. Two of the hybrid lines contained human X chromosomal material derived from different adult cell types; the third line contained the X plus three other chromosomes from a human testicular teratocarcinoma. These hybrids therefore provide us with a system to investigate the ability of an embryonic genome to regulate expression of genes contained on various human chromosomes and may allow investigation of human genes important in embryonic development. MATERIALS AND METHODS Cell lines. Table 1 lists the series of human-mouse somatic cell hybrids constructed by using PCC4AO, a clone of the EC line PCC4 (29), as the mouse parent. The PCC4AO line (henceforth referred to as PCC4) has a near diploid XO karyotype. Two of the hybrid lines. MCP6 and GEOC4, were made by the microcell fusion technique (15), whereas human-mouse hybrid cell lines (Table 1) served as the human chromosome donors since human cells do not readily undergo microenucleation. Briefly, the human-mouse hybrid parent line was cultured for 3 days in the presence of colchicine (0.2 ,ug/ml). Enucleation was achieved on discontinuous Ficoll gradients supplemented with cytochalasin B at 10 ,ug/ml. Microcells were pooled and fused with 6 x 106 PCC4 cells, using inactivated Sendai virus as described before (34). Isozyme and karyotype analyses have shown that MCP6 contains an X/6 translocation chromosome as its only human genetic component (19) which is maintained in the hybrid by hypoxanthine-amethopterin-thymidine selective medium. GEOC4 contains approximately twothirds of the long arm of the human X chromosome translocated onto a mouse chromosome; the short arm of the X has been lost from the hybrid as detected karyotypically by G1l staining (10) and by the absence of the short arm-coded 12E7 antigen expression. The third hybrid, PC21A1, was obtained by conventional polyethylene glycol fusion of PCC4 with the human testicular teratocarcinoma-derived line 2102Ep (3). This hybrid contains a whole human X chromosome and in addition retains human chromosomes 11, 15, and 17 as determined karyotypically. Cell culture. All cell lines were cultured in Dulbecco-modified Eagle medium (DME) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and nonessential amino acids. Media for the hybrid lines also contained HAT (hypoxanthine at 100 ,uM, amethopterin at 10 mM, thymidine at 16 ,uM). For differentiation induction, cells were adapted to grow in the presence of retinoic acid (10-8 M) (M. Sherman, personal communication). Cells were exposed to 10-8 M retinoic acid for 48-h periods and then left to recover for 48 h. After 6 to 10 such changes, cells remained healthy in the presence of 108 M retinoic acid while retaining their EC phenotype. Once adaptation had occurred, it was not necessary to repeat it. Adapted cells then exposed to 10-6 M retinoic acid showed marked changes consistent with differentiation.

FACS analysis. Cells for fluorescence-activated cell

HYBRIDS

2261

sorter (FACS) analysis were prepared and used as described before (2). In assays with the antibody Chrome-1, cells were first fixed and permeablized with 50% ice-cold ethanol. Cells were incubated for 1 h at 4°C with saturating concentrations of first antibody. Cells were washed and incubated for a further 1 h at 4°C with fluorescein isothiocyanate-labeled rabbit anti-mouse immunoglobulin G1 (IgGl). Cells were washed and analyzed on an FACS type I. Immunoprecipitation. Each cell line was seeded at 106 cells per 50-mm plate with 1 ml of DME-HT medium (minus methionine) containing 10% dialyzed fetal calf serum, nonessential amino acids, and 200 ,uCi of [35S]methionine per mmol and incubated for 16 h. Media were collected and precipitated with 10% polyethylene glycol. The precipitates were redissolved and immunoprecipitated with anti-laminin, anti-type IV procollagen, or control antiserum, using fixed Stap/zvlococcius alureuis. The immunoprecipitates were analyzed by 10% sodium dodecyl sulfate (SDS) gel electrophoresis after reduction with ,-mercaptoethanol (38). Indirect radioimmunoassay. Radioimmunoassay was performed with, as second antibody, 1251-tagged F(ab)'2 rabbit anti-mouse immunoglobulin (ca. 30 RCi/ ,Lg), as previously described (18). Antibodies. Monoclonal antibodies were as follows: (i) monomorphic anti-HLA-A, -HLA-B, and -HLA-C. W6/32 (6); (ii) anti-H-2D*, B22.242.129 (40), donated by G. Hammerling; (iii) anti-SSEA-1 (59), donated by D. Solter; (iv) control P3-X63.Ag8 immunoglobulin (36), used as a control for nonspecific binding; (v) anticytokeratin Chrome-1 (8). All antibodies were used at saturating concentrations. Antisera. The following antisera were used. (i) Rabbit antiserum raised against mouse laminin (12) was donated by B. Hogan. The antiserum was absorbed with fibronectin before use. (ii) Goat antiserum raised against type IV procollagen by G. Martin was provided by B. Hogan. Plasminogen activator. Cells were seeded at 2 x 106 per 50-mm plate and incubated with 2 ml of DME supplemented with 0.5 mg of bovine serum albumin, TABLE 2. Expression of SSEA-1 and H-2h in PCC4human hybrids cpm bound (SD)' Negative Hybrid Anti-H-2b Anti-SSEA-1 X63. Ag8) (B22.22.129)(P3.control

A122-4-19

421 (65) 839 (123) 544 (180) 1.588 (257) 421 (65) 462 (92) 309 (5) 437 (118) 282 (64) 292 (32) 6,573 (607) pGlgb " The antibody binding was determined by indirect radioimmunoassay. The values represent the means results of three determinations. Cells were scored as positive when they bound at least three times more than the background observed in the binding of P3.X63.Ag8. Positive results are shown in boldface type. GEOC4 was borderline positive for H-2. b The H-2 -positive melanoma cell line PG19 (32) was used as positive control for H-2" expression.

MCP6 GEOC4 PC21A1 PCC4AO

3,732 8,257 3,861 4,271

(34) (442) (610) (375)

2262

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FIG. 1. Morphology of GEOC4 EC cultures and differentiated cloned derivatives MP2. MP1I and GPC from retinoic acid-treated cultures of thc hybrids (see Table 1 for derivation and phenotype). nonessential amino acids. hypoxanthine. and thymidine for approximately 16 h. Conditioned media were collected and centrifuged to remove any cells and then concentrated fivefold with an Amicon macrosolute concentrator. Conditioned media were separated on an 11% SDS-polyacrylamide gel (38) containing copolymerized gelatin: duplicate gels were run with and without plasminogen (0.01 U/ml. Sigma Chemical Co.). Bands of plasminogen activator activity were visualized as described before (21). Alkaline phosphatase. Alkaline phosphatase activity was determined by histochemical staining of fixed monolayers of cells (7). Malic enzyme. Malic enzyme present in cell extracts was separated by starch gel electrophoresis, and isozymes were visualized as previously described (55).

RESULTS Table 1 summarizes the construction of the three human-mouse hybrid lines which have EC line PCC4 as the mouse parent.

The phenotype of the hybrid lines which contain either a single or a few human chromosomes was investigated initially by examination of morphology and cell surface antigen profile. The lines all show a conventional EC morphology. Indirect radioimmune assay demonstrated that the three lines all express the monoclonal antibody-defined cell surface antigen SSEA-1, but express little or no H-2 class I antigens (Table 2). This antigenic phenotype is typical of murine EC cells and of preimplantation-stage embryos (59). The borderline positive result for H-2 on GEOC4 indicates that these cultures may contain small subpopulations of differentiated cells, a phenomenon found with several murine EC cultures (43). Induction of differentiation. When the retinoic acid-adapted PCC4 cells and the three hybrid cultures were grown in the presence of 10-6 M retinoic acid, a change in morphology occurred

VOL. 3, 1983

DIFFERENTIATION OF TERATOCARCINOMA HYBRIDS

2263

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over the first 24 to 72 h. The majority of the cells lost their typical EC morphology and "flattened out." During this time, some cell death occurred in the hybrid cultures. Continued culture in retinoic acid resulted in the appearance of a variety of apparently different morphological cell types, including endoderm, epithelial, and fibroblast-like. The predominance of any particular cell type was related to cell density and duration of retinoic acid treatment. Differentiated cell lines. Dominant cell types from the retinoic acid-treated cultures of the three hybrids were isolated by cloning after various times of exposure to retinoic acid. The cloned lines (Table 1) have morphologies resembling either the murine parietal endoderm line PYS (39) or fibroblastoid cells (Fig. 1). These lines grow and maintain their differentiated morphology and phenotype (as described below) without the presence of retinoic acid. The human chromosomal components are maintained

in the MCP6 and GEOC4 differentiated derivatives and are identical to those in the original EC cells as far as can be detected karyotypically. The PC21A1 differentiated derivatives contain a whole human X (two lines) or a short armdeleted X chromosome (one line) but have lost the other nonselected human genetic material. Antigenic phenotype. Culture in the presence of retinoic acid brought about a rapid decrease in the expression of the embryonic cell surface antigen SSEA-1 in PCC4 and the three hybrid lines as detected by immunofluorescence, using the FACS (Fig. 2). The percentage of cells expressing SSEA-1 decreased to under 10% during an 8- to 9-day period in cultures of PCC4, MCP6, and GEOC4; a similar decrease of SSEA-1 occurred within 2 to 3 days in cultures of PC21A1. Longer-term cultures tended to show a subsequent small increase in the proportion of SSEA-1-positive cells, presumably due to the outgrowth of remaining EC cells which failed

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FIG. 2. Time course showing extinction of SSEA-1 expression after culture of GEOC4 in the pre sence of retinoic acid (10-6 M), analyzed by using th4 e FACS. Closed symbols show PCC4 (0) and PC21A1 I(A) Cells incubated with anti-SSEA-1; corresponding o0 pen symbols show cells incubated with negative control immunoglobulin P3.X63.Ag8. to differentiate (60). The cloned derivattives of the EC hybrids were all SSEA-1 negatiNve.

Expression of the class I antigens of thie major histocompatibility complex (H-2b halplotype) was not detected on the surface of retinc ic acidtreated cultures of PCC4 or the hybrids e ither by indirect radioimmunoassay or by FAC'S analysis. This result is somewhat surprisinig since other groups have demonstrated the prersence of H-2-positive cells after differentiation of EC cultures (35), and it may reflect differe.nces in developmental potential between the var ious EC lines used. In addition, surface H-2 exj pression was not detected on any of the differientiated derivatives of the hybrids, a result co insistent with the lack of detection in differentiatirig PCC4 cells. Differentiation of EC cells has been slhown to be associated with the appearance of inltermediate filaments which react with anti-cyt okeratin antibodies (33, 53). Chrome-1, a receintly derived monoclonal antibody which reaccts with cytokeratin filaments (8) is therefore;a useful marker of EC differentiation. Ethanol-fil ed cells from the PCC4 and hybrid cultures were stained with Chrome-1 in an immunofluorescen(ce assay and analyzed on the FACS (Table 3). LJndifferentiated cultures all showed a very Ilow, although variable, proportion of Chrome1-1 -positive cells. The differentiated derivat:ives all showed a much higher proportion of Ch rome-lexpressing cells, with the parietal eridoderm lines having the highest levels. In tI his they resemble the high-level-expressing pariietal en-

doderm line PYS. The fibroblast-like clone MP1 showed a variable intermediate level of Chrome1-positive cells. Plasminogen activator. Tissue plasminogen activator is a plasminogen-dependent protease which is first synthesized in vivo in the murine parietal endoderm as it differentiates from the inner cell mass (62). During in vitro differentiation of EC cultures, an enhanced secretion of tissue plasminogen activator occurs (31, 58, 61). Conditioned media from PCC4 and the hybrids were assayed for plasminogen-dependent proteolysis after separation on SDS-polyacrylamide gels (Fig. 3). The control parietal endoderm line PYS showed a single major plasminogen-dependent band of activity at 86,000 molecular weight (86K).

A weak band of

independent

nonspecific plasminogenactivity

comigrated

this 86K band in the PYS media

as

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as

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media from all the other cell lines examined; however, addition of the plasminogen substrate gave a much stronger band of activity at 86K in the PYS and the parietal endoderm derivative MP2 media. Cell lines with nonparietal endoderm phenotypes (PCC4, MCP6, and MP1) did not show increased activity of the 86K band when plasminogen was added, suggesting that in these lines the activity at this molecular weight was mainly due to a nonspecific esterase, although the presence of a small amount of plasminogen-dependent activity at 86K in the EC cells and the MP1 line cannot be ruled out. A weaker plasminogen-dependent band of 58K was present in the cell cultures which did not show the 86K band (PCC4, MCP6, and MP1). This band, which is different from the tissue TABLE 3. Reaction of hybrid cell lines with

an

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Control PCC4AO PYS

PSASEb

Phenotype

Proportion of Chrome-1positive cells"

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6 83 25

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form of plasminogen activator, may represent one of the various other low-molecular-weight forms found in embryonic tissues (41). A gene which codes for one form of human plasminogen activator has been provisionally mapped to chromosome 6 (37). To investigate whether a possible human contribution to the tissue plasminogen activator activity could be detected in the parietal endoderm derivative of MCP6, called MP2, a revertant line was derived from MP2 which no longer contains any of the X/6 human chromosome. However, when conditioned medium from the revertant MP2 culture was compared with MP2 medium, no differences in plasminogen activator activity patterns were detected. High-molecular-weight glycoproteins. Highmolecular-weight glycoproteins characteristic of the parietal endoderm are also synthesized and secreted by some differentiated EC cells (22, 24, 64). By using specific antisera against type IV procollagen and laminin to immunoprecipitate these glycoproteins from [35S]methionine-labeled conditioned medium, new or greatly enhanced synthesis and secretion of laminin and type IV procollagen was detected in retinoic acid-treated cultures of PCC4 cells and the hybrids (Fig. 4). The parietal endoderm-like derivatives of the hybrids secreted copious amounts of both of these proteins, equivalent to levels in the parietal endoderm line PYS, whereas their EC progenitors were found to secrete only trace amounts of laminin. The fibroblast-like MP1 line

2265

secreted both laminin and type IV procollagen but at a considerably lower level. Alkaline phosphatase. Alkaline phosphatase activity has been used extensively as a marker for early embryonic mouse cells as well as EC cells. Upon differentiation in vivo and in vitro, alkaline phosphatase drops to low or negligible levels (9, 64). Histochemical staining showed that all of the cells in cultures of PCC4 and the undifferentiated hybrids had high levels of alkaline phosphatase activity. In contrast, most of the cells in the retinoic acid-treated cultures and the differentiated derivatives showed no activity. A few weakly positive cells were observed in the latter cultures. Tumor formation. The three EC hybrids and two of the differentiated derivatives were tested for their ability to form tumors in nude mice after subcutaneous injection of 107 cells per site. The EC hybrids MCP6, GEOC4, and PC21A1 formed tumors efficiently at all sites injected (minimum of four sites per cell line), and histological examination showed that these tumors resembled those formed by the parent PCC4 line in that they consisted mainly of EC cells. The parietal endoderm hybrid line GPC11 formed tumors in two out of four sites injected, and the tumors showed a characteristically large amount of extracellular matrix. The MP1 line (fibroblastlike morphology) formed angiosarcomas in two out of three sites injected. The human form of the X-linked enzyme glucose-6-phosphate dehydrogenase was found in all tumors examined, indicating that the tumor tissue was derived from the injected cells and that a substantial proportion of the cell population retained the human X chromosome. Malic enzyme. It has previously been shown that in hybrid MCP6 cells expression of a human gene product, malic enzyme (EC 1.1.1.40), was altered by being in the mouse teratocarcinoma background (55). Briefly, the human lymphoid line parent G3.32.2, from which the X/6 translocation chromosome originated, was totally deficient in malic enzyme, an enzyme mapped to the long arm of chromosome 6. This deficiency is a characteristic found in all lymphoid lines derived from Burkitt's lymphomas (55). In the hybrid cell line GIR6, made between G3.32.2 and the mouse line 1R (Table 1) and, in other humanrodent hybrids, made with G3.32.2 as the parent, the human malic enzyme was still not expressed, and the deficiency behaved as though it were the result of a deletion. However, in MCP6, made by microcell fusion between GIR6 and PCC4, malic enzyme was found to be present at normal cultured cell levels, and the activities of the mouse and human isozymes were approximately equivalent. Human malic enzyme was also found in an independent hybrid

2266

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line, PCG (55), made by conventional fusing of G3.32.2 with PCC4, supporting the evidence that the teratocarcinoma cell has the ability to selectively reactivate the human malic enzyme gene locus. Analysis by starch gel electrophoresis of malic enzyme isozymes in retinoic acid-treated cultures of MCP6 and in the differentiated derivatives MP1 and MP2 indicated that the activity of the human isozyme was very much reduced relative to that of the mouse isozyme (Fig. 5), implying that there is a specific decrease in human malic enzyme expression (relative to mouse) consequent upon differentiation. DISCUSSION As summarized in Table 4, the three hybrid cell lines constructed by introduction of a limited amount of human chromosomal material into

1

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FIG. 5. Malic enzyme in the undifferentiated and differentiated cultures of the MCP6 hybrid as separated by starch gel electrophoresis. Lanes: 1, human control; 2, PCC4; 3, MCP6 undifferentiated; 4, MCP6 with 7 days of retinoic acid treatment.

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TABLE 4. Summary of phenotypic characteristics of PCC4-human hybrids and their differentiated derivatives

(cytokeratin)

Laminin and type procollagen

+++

-

-

Retinoic acid-Treated mass Cultures

+

+

+

Differentiated clones

-

+

+ +

-

+ (35%)

+

Cell line

EC

PCC4 MCP6 GEOC4 PC21A1

SSEA-1

Chrome-1 antigen

Plasminogen activator (tissue form)

phosphatase

+

+

Alkaline

+++ ++

GPC MP2 PP1

MP1

the mouse EC cell line retain all the phenotypic properties of EC cells examined, which includes morphology, ability to differentiate into a variety of cell types in the presence of retinoic acid, SSEA-1 surface antigen expression, lack of cell surface H-2 and Chrome-1 (anti-cytokeratin) reactivities, low or negligible secretion of tissue plasminogen activator and the glycoproteins laminin and type IV collagen, and high alkaline phosphatase activity. The hybrids undergo retinoic acid-induced differentiation which is similar to that of PCC4 in our hands, and cloned constitutively differentiated lines have been isolated from all three hybrids which have phenotypes closely resembling either parietal endoderm (22, 24, 58, 63, 64) or an as yet undetermined but morphologically fibroblast-like phenotype. A similar cell line has been derived recently from the EC line PCC3 (51). For MCP6 and GEOC4, the differentiated derivatives are identical genetically with the EC progenitor, at least on a gross chromosome level. The finding that these hybrids behave as EC cells allows us to investigate the extent to which the embryonic genome can reprogram according to its own developmental program expression from chromosomes which had progressed through various stages of development. It was found that the EC cell background did direct the reexpression of a previously absent human enzyme, malic enzyme, and although the developmental significance and the mechanism of this reexpression are unclear, the effect appears to be specific to the EC cell background. To determine whether certain genes which are expressed in the differentiated human parental cells but whose homologs are not normally expressed in

-

+

EC cells are turned off in the hybrids, we are currently analyzing the expression of HLA class I and class II antigens in the MCP6 hybrid, which contains the chromosome 6-coded major histocompatibility complex. Results indicate that class I heavy chain genes continue to be transcribed (although the antigens are not expressed on the cell surface because the light chain subunit, I32 microglobulin, is not present [5, 19], whereas class I transcription is turned off completely (Benham et al., EMBO J., in press). Thus, some types of genes may be susceptible to regulation by the embryonic program, whereas others may not, and the expression of these and other genes is being investigated further. These hybrids can also be used to ask the opposite question, which is whether genes not expressed in the differentiated parent cells but normally turned on during early embryonic differentiation can be reexpressed by replacing them into an embryonic environment. We are attempting to address this question by looking at the expression in the hybrids of human genes homologous to mouse genes known to be developmentally regulated, for example, plasminogen activator, a gene which is reported to be localized to chromosome 6 in humans (37), and by

identifying developmentally regulated X chromosome-coded genes. The techniques of somatic cell genetics have previously been used to study the control of early development and differentiation. Mousemouse hybrids made between EC cells and various differentiated cells retained an overall EC phenotype when thymocytes (4, 47, 57) and, in some cases, splenocytes (17) and erythroleukemic cells (48) are the fusion partner. In con-

2268


trast, in other fusions, hybrids showed differentiated phenotypes when the fusion partners were embryonic fibroblasts (48), L cells (4, 14, 30), erythroleukemic cells (46), or parietal endoderm cells (25). In these intraspecific fusions, it was possible to distinguish between the genetic contribution from the two parental cell types only at a few available polymorphic loci, so the expression pattern of only a very limited number of genes could be analyzed. Introduction of a selectable human chromosome into a mouse EC cell has the advantage that a great many more gene products can be distinguished, and the contribution from the two genomes can be analyzed in more detail. In addition, a single introduced chromosome may exert less dominant disturbance of the EC program than the presence of several differentiated cell chromosomes. A possible disadvantage of introducing human genetic material into a mouse EC cell is that it may not respond to the mouse developmental control signals. However, appropriate developmental changes in human gene expression have been seen in two other rodent-human hybrid systems in vitro. In the first, dimethyl sulfoxide was able to induce the synthesis of human ox and y globulin mRNA in hybrids made between mouse erythroleukemic cells and human fibroblasts (65). Secondly, a somatic cell hybrid made by fusing a rat muscle cell line with human fetal muscle cells was induced to form myotubules by manipulation of culture conditions. Isozyme analysis showed that both mouse and human muscle-specific gene products were newly expressed as the differentiation progressed (56). Thus, the EC hybrids described in this report may provide a system for identifying human genes expressed during embryonic development and elucidating the molecular mechanisms by which they are regulated. ACKNOWLEDGMENTS We thank Brigid Hogan for supplying antisera: Peter Andrews, Davor Solter. and Ivan Damjanov for examination of

the tumors from nude mice, Ellen Solomon and Bengt Bengtsson for help with chromosome analysis, and Sue Povey and Dallas Swallow for isozyme analysis. We thank George Banting, Mohamad Parkar, Sue Chambers. Karina Stanley, and Laura Davis for technical assistance. LITERATURE CITED 1. Adamson, E. D., M. T. Evans, and G. G. Magrane. 1974. Biochemical markers of the progress of differentiation in

cloned teratocarcinoma cell lines. Eur. J. Biochem. 79:607-615. 2. Andrews, P. W. 1983. The characteristics of cell lines derived from germ cell tumors. In I. Damjanov. B. B. Knowles. and D. Solter (ed.). Human teratomas. Humana

Press, Clifton, N.J. 3. Andrews, P. W., D. L. Bronson, F. J. Benham, S. Strickland, and B. B. Knowles. 1980. A comparative study of

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10.

11. 12.

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