Nullipotent Teratocarcinoma Cells Acquire the Pluripotency for Differentiation by Fusion with Somatic Cells

Differentiation

Differentiation (1982) 23: 83-86

(

Springer-Verlag 1982

Nullipotent Teratocarcinoma Cells Acquire the Pluripotency for Differentiation by Fusion with Somatic Cells Tadao Atsumi’, Yasuaki Shirayoshi, Masatoshi Takeichi and T.S.Okada Department of Biophysics, Faculty of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606,Japan

Abstract. By fusion of nullipotent embryonal carcinoma F9 cells with certain somatic cells freshly collected from differentiated tissues such as thymus and lens, pluripotent hybrid cell lines were obtained. They exhibited a wide spectrum of differentiation, including neural tubes, cartilages, skeletal muscles, ciliated epithelia and others, in solid tumors formed after injection into syngeneic mice. Cells from these tumors differentiated into several cell types when cultured in vitro. A possibility of the introduction of genes to code the factors for regulating differentiation into F9 cells by fusion is suggested.

Introduction F9 cells are nullipotent embryonal carcinoma cells isolated from pluripotent OTT6050 cells [l]. Primitive endodermal cells are only possible products of differentiation of F9 cells, which are induced spontaneously or after treatment with retinoic acid [5, 11, 121. No other means are known to elicit major differentiations from F9 cells. It is known that the differentiation capacity of pluripotent teratocarcinoma cells is often influenced by fusion with somatic cells, and the capacity is often lost in hybrid cells [6]. A reciprocal case, i.e., an acquisition of the differentiation capacity of nullipotent F9 cells by cell fusion, will be reported here. We describe the properties of several pluripotent hybrid cells, which are capable of differentiation into a variety of cell types. These were obtained by means of cell fusion of F9 cells with certain somatic cells freshly collected from differentiated tissues or from their primary cultures.

for cell fusion. The following three hybridizations were carried out: (a) OTF9-63 cells x C3H/HeJ prenatal (18 days post coitum) mouse thymocytes (hybrid cells being designated as F9T), (b) OTF9-63 cells x C3H/HeJ adult mouse lens epithelial cells (F9Le/C3H) and (c) OTF9-63 cells x 129/Sv prenatal (18 days post coitum) mouse lens epithelial cells (F9Le/l29). Thymocytes were freshly collected from thymus. Lens epithelial cells isolated by a method of Hamada and Okada [4] were cultured in Dulbecco’s modified Eagle’s minimal essential medium supplemented with 10% fetal calf serum (DMEM-1OFS) for 2 weeks before cell hybridization. Somatic cell hybrids were produced by incubating the mixture of parent cells in 50% polyethylenglycol (M.W. 1500, Wako chemicals, Japan) as described by Davidson and Gerald [2]. Hybrid cells were selected from their parent cells in the medium (DMEM10FS) containing 4 x lo-’ M aminopterin, 1 x M hypoxanthine, 1.6 x M thymidine (HAT) and 4x M ouabain. After 2 or 3 weeks, growing colonies became microscopically detectable. Each colony was collected and transferred into a respective culture dish with the HAT-ouabain selection medium. Electrophoresis for Detecting the Isozyme Pattern Cellulose acetate gel (Cellulogel, Chemetron) electrophoresis was carried out to detect the isozyme pattern of glucosephosphate isomerase (GPI;EC 5.3.1.9) according to the method described by Eppig et al. [3]. About 1 x lo6 washed cells were homogenized by freeze-thawing in 100 $50 mM Tris-HC1, 1 mM EDTA and 1 mM /l-mercaptoethanol at pH 7.5 and the supernatants obtained after centrifugation was applied onto slots made in the gels (1-2 pl/slot). Elec-

Methods Cell Culture and Fusion Hypoxanthine phosphoribosyltransferase-deficient and ouabain-resistant F9 cell line (OTF9-63), isolated by Rosenstraus et al. [8, 91 and supplied by Dr. K. Sekikawa (Sapporo Medical College), was used as parent F9 cells Please address correspondence to : Masatoshi Takeichi, Department of Biophysics, Faculty of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606, Japan 1 Present Address : Cancer Institute, Kami-ikebukuro, Toshimaku, Tokyo 170, Japan

Fig. 1. Electrophoresis of lysates of hybrid and control cells. OTF963 parental cells (129/Sv strain) show only GPI-AA isozyme band, whereas both C3H/HeJ thymocytes and C3H/HeJ lens epithelial cells produce only GPI-BB. All hybrid cells between OTF9-63 and C3H/HeJ cells produce not only GPI-AA and GPI-BB but also a hybrid band, GPI-AB. A mixture of lysates of OTF9-63 cells and thymocytes does not produce the hybrid band. 1, OTF9-63; 2, thymocytes; 3, F9T/4; 4, F9T/17; 5, F9T/30; 6, F9Le/C3H/2; 7, F9Le/C3H/4; 8, F9Le/C3H/l8; 9, lens epithelial cells; 20, a mixture of OTF9-63 and thymocytes

0301-4681/82/0023/0083/$01.OO

a4

Fig. 2a, b. Karyotypes of OTF9-63 (a) and hybrid F9Le/C3H/4 cells (b). Arrows pointing to metacentric marker chromosome characteristics of OTF9-63 parcntal cells. x 1.1 90

a)

121

* 0

Tablc 1. Differentiation spectrum of hybrid cclls after intraperitoneal injection

30

40

50

60

70

BO

L

Q)

Cloncs

Differcntiation in solid tumors

Differentiation in culture of tumors'

F9T/4

Neural tubes

F9T/17

Tubular structures

F9T/30

Neural tubes

F9Le/C3H/2

Neural tubes

F9Lc/C3H/4

Neural tubes, cartilages, tubular structures with ciliated epithclium Neural tubes, cartilages

Neuronal cells, fibroblasts, epithelial cells" Fibroblasts, epithelial cells Neuronal cells, fibroblasts, epithelial cells Neuronal cells, fibroblasts, epithelial cells Fibroblasts, epithelial cells

n E a

z

F9Le/C3H/18

30

40

Number

50

of

60

70

ao

Chromosomes

Fig. 3. Histogram of chromosome number. a OTF9-63; b F9Le/ C3H/4

trophoresis was carried out in 0.043 M Tris, 0.046 M glycine buffer, pH 8.6, at 180 V for 2 h. Chromosomal Analysis

Dividing cells in cultures were harvested after 4 h treatment with 0.01 mg/ml colcemid. They were swollen in 1% sodium citrate for IS min at 37" C and fixed in methanol-glacial acetic acid (3:l). Cells were then spread on slides glasses, air-dried and stained with Giemsa for counting chromosome number.

F9Le/l29/9

Neural tubes, cartilages, muscles

Neuronal cells, fibroblasts, epithelial cells Neuronal cells, fibroblasts, epithelial cells, beating cardiac myocytes

Cells derived from tumores were cultured in the H AT-ouabain selection medium as described in the text and periodically observed by an inverted phasecontrast microscope Ten subclones were isolated from cultures of this clone, of which, two were pluripotent, while the others were nullipotent Probably an endodermal derivative

Detection of Differentiation in vivo and in vitro

To examine the differentiation potential, 5 x lo6 cells of each hybrid clone were i.p. injected into syngeneic mice; F9T and F9Le/C3H cells into (C3H/HeJ x 129/Sv) F1 mice,

85

Fig.4a-d. Differentiation in tumors of hybrid cells. a, b and c show the differentiated structures found in histological sections of solid tumors produced in syngeneic host mice 20-30 days after injection of hybrid cells. a the differentiation of a tubular structure with ciliated epithelium (7)from F9Le/C3H/4. b the differentiation of cartilage (CA) and tubular structures from F9Le/C3H/l8. c the differentiation of striated muscles from F9L,e/l29/9. d a phase-contrast micrograph of 7-day culture of cells derived from solid tumors formed 20-30 days after the injection of F9T/4 cells. Note the differentiation of a clump of neuronal cells with axon-likc processes. a, x 240; b, x 120; c, x 260; d, x 150

whereas F9Le/129 cells into 129/Sv mice. After 20 or 30 days most hybrid clones produced solid tumors. They were cut into two pieces, one fixed for histological observations and the other dissociated with 0.05% trypsin in Caz+-and MgZ+-free saline. The trypsinized cells were cultured in Falcon culture dishes with the HAT-ouabain selection medium. Results and Discussion

So far 66 clonal hybrid cell lines were isolated and their differentiation potential was examined. The hybrid nature of F9T and F9Le/C3H cells was confirmed by the heterodimeric banding pattern for GPI after electrophoresis of cell lysate of each hybrid clone (Fig. 1). F9Le/l29 cells were assumed to be hybrids because of their resistance against both HAT and ouabain. Chromosomal analysis also supports the hybrid nature of cell lines obtained by cell fusion. Normal mouse thymocytes and lens epithelial cells contain the diploid number of 40 chromosomes. OTF9-63 cells have 38-40 chromosomes and contain a single metacentric marker chromosomes [9]. A line of OTF9-63 cells used for the present experiment has a mode of 42 chromosomes, a range of 38-45, containing one metacentric chromosome (Figs. 2 and 3). The expected modal chromosome number in hybrids would be about 80. Hybrid F9Le/C3H/4 retained about 70-80 chromosomes. The range was 55-78 per mode (Fig. 3). The chromosome number of hybrid cells was

nearly the sum of the parental chromosome number. In addition, F9Le/C3H/4 cells contained a single metacentric chromosome (Fig. 2). Other hybrid clones were similar to F9Le/C3H/4 in the number of chromosomes and all contained one metacentric marker chromosome. Of these, 7 clonal lines, three of 36 F9T clones, three of 20 F9Le/C3H clones and one of 10F9Le/129 clones, exhibited a variety of differentiations in solid tumors formed after the injection into syngeneic mice (Table 1 ; see also Figs. 4a, b and c). The hybrid nature of these tumors with differentiated tissues derived from F9T and F9Le/C3H cells was confirmed by the isozyme pattern of GPI after electrophoresis of the supernatant of their homogenates. As listed in Table 1, structures derived from all three germ layers in situ were recognized in tumors of the hybrid cells. After in vitro culture of these tumor cells, various types of differentiated cells appeared. Of these the differentiation of neuron-like cells with very elongated processes was common in most clones (Fig. 4d). In one clone, beating cardiac myocytes were observed (Table 1). Since these cultures were made with the HAT-ouabain selection medium, it is assumed that all the differentiated cells are derived from the hybrid clones, but not from the tissues of host animals included in the tumors. We also produced 30 hybrid clones by fusion of OTF963 cells with mouse L cells [A and 28 hybrid clones by fusion with hepatoma cells [lo]. All these hybrid cells were nullipotent after injection into syngeneic hosts. Injection of 5 hybrid clones F9 x F9 did not cause tumors containing any differentiated tissues.

86

It was known that F9 cells can only differentiate into primitive endoderm [5, 11, 121. Parental primary cells chosen to be fused with F9 cells in the present work were thymocytes and lens epithelial cells. Thymocytes are of mesodermal origin, whereas lens epithelial cells are of ectoderma1 origin. Irrespective of such differences in the germ layer origins, both were effective, by fusion with F9 cells, in inducing pluripotent hybrid lines, which can differentiate into structures to be derived from all three germ layers. Our results can be interpreted to indicate that nullipotent embryonal carcinoma cells are deficient in the factor(s) necessary for regulating somatic cell differentiation. Genes to code for such factors must be introduced from the differentiated cells into embryonal carcinoma cells by cell fusion, and thus some hybrid cells gain a potential for pluripotent differentiation. Our results also suggest that some of the established cell lines are unable to express such genes when fused with embryonal carcinoma cells. Acknowledgements. We thank Dr. K. Sekikawa for providing us with OTF9-63 cells, Mr. K . Agata for advice and assistance in the preparation of lens epithelial cells and Dr. H. Kondoh for his valuable suggestions. This work was supported by the grant for Basic Cancer Research from the Japan Ministry of Education, Science and Culture to T.S. Okada. We also thank Mrs. Y.Yonesaki for help in the preparation of the manuscript.

References 1. Bernstine EG, Hooper ML, Grandchamp S , Ephrussi B (1973)

Alkaline phosphatase activity in mouse teratoma. Proc Natl Acad Sci USA 70:3899 2. Ddvidson RL, Gerald PS (1977) Induction of mammalian somatic cell hybridization by polyethylene glycol. In: Prescott

DM (ed) Methods in cell biology, vol. 15. Academic Press, New York and London, p 325 3. Eppig JJ, Kozak LP, Eicher EM, Stevens LC (1977) Ovarian leratomas in mice are derived from oocytes that have completed the first meiotic division. Nature 269:517 4. Hamada Y, Okada TS (1977) The differentiating ability of rat lens epithelial cells in cell culture. Develop Growth Differ 19:265 5. Hogan BLM, Taylor A (1981) Cell interactions modulate embryonal carcinoma cell differentiation into parietal or visceral endoderm. Nature 291 :235 6. Mcburney MW, Strutt B (1979) Fusion of embryonal carcinoma cells to fibroblast cells, cytoplasts, and karyoplasts. Developmental properties of viable fusion products. Exptl Cell Res 124:171 7. Murdyama-Okabayashi F, Okadd Y, Tachibana T (1971) A series of hybrid cells containing different ratios of parental chromosomes formed by two steps of artificial fusion. Proc Natl Acad Sci USA 68: 38 8. Rosenstraus MJ, Levine AJ (1979) Alterations in the developmental potential of embryonal carcinoma cells in mixed aggregates of nullipotent and pluripotent cells. Cell 17:337 9. Rosenstraus MJ, B a h t RF. Levinc AJ (1980) Pluripotency of somatic cell hybrids between nullipotent and pluripotent embryonal carcinoma cells. Somat Cell Genet 6:555 10. Sato H, Belkin M. Essner E (1956) Expcrimcnts on an ascilcs hepatoma. 111. The conversion of mouse hepatomas into the ascites form. J Natl Cancer lnst 17 :1 11. Sherman MI, Miller RA (1978) F9 embryonal carcinoma cells can differentiate into cndodenn-like cells. Dev Biol 63: 27 12. Strickland S, Mahdavi V (1978) The induction ofdifferentiation in teratocarcinoma stem cells by retinoic acid. Cell 15:393

Received June 1982 / Accepted in revised form August 1982