cloned human teratoma cells differentiate into … · cloned human teratoma cells differentiate...

J. Cell Sd. 72, 37-64 (1984) 37Printed in Great Britain © The Company of Biologists Limited 1984

CLONED HUMAN TERATOMA CELLSDIFFERENTIATE INTO NEURON-LIKE CELLS ANDOTHER CELL TYPES IN RETINOIC ACID

S. THOMPSON1, P. L. STERN1, M.WEBB2, F. S. WALSH2,W. ENGSTROM1, E. P. EVANS3, W.-K. SHI1, B. HOPKINS1 ANDC. F. GRAHAM1

1 Department of Zoology, South Parks Rd, Oxford 0X1 3PS, UX.2 Institute of Neurology, Queen Square, London WC12 3BG, U.K.3 Dunn School of Pathology, South Parks Rd, Oxford 0X1 3RE, U.K.

SUMMARY

Single cell clones were isolated from the human teratoma line, Tera-2. The cells of three of theseclones were studied. The progressively growing cells were shown to be tumorigenic, and they werecharacterized by the lack of expression of /32-microglobulin and HLA-A,B,C determinants on thecell surface. The majority of the cells expressed Thy-1 antigen and a 90 X 103 molecular weightprotein recognized by the monoclonal antibody F10.44.2; between a third and half of the cellsexpressed the sugar specificities detected by the anti-SSEA-1 monoclonal antibody. In response to5 X 10~5 M-retinoic acid applied to cells in monolayer culture, the cells differentiated into a popula-tion of flat static cells arrested in the G\ phase of the cell cycle. A substantial proportion of thesedifferentiated cells expressed /32-microglobulin and 43 X 103 molecular weight HLA-A,B,Cpolypeptides, Thy-1, SSEA-1 sugar determinants, and the 90 X lC^Mr protein recognized byF10.44.2. The apparent molecular weight of fibronectin secreted by the cells decreased by about5 X 103Afr to 235 X 103M, after differentiation. The progressively growing cells lacked reactivitywith reagents that mark cells in the nervous system. Following aggregation and retinoic acid treat-ment, neuron-like cells were formed. These cells reacted with reagents that also react with humanneurons in culture: they reacted with tetanus toxin, the anti-neurofilament antibodies BF10 andRT97, the anti-ganglioside, GQlc antibody F12 A2B5, and anti-Thy-1.

The progressively growing cells of these Tera-2 clones are therefore capable of forming at leasttwo types of cell: the flat cells in monolayer cultures and the neuron-like cells. None of the cellpopulations reacted with the monoclonal antibody against SSEA-3 and these cloned cells aretherefore distinct from previous isolates from Tera-2.

INTRODUCTION

If all the cell types seen in human teratomas are derived from a single multipotential

stem cell, then that cell would be recognized by its ability to form those cell types.

Human testicular tumours frequently contain embryonal carcinoma, seminoma, and

cells that resemble those found in extra-embryonic foetal tissues, for example placen-

tal trophoblast and yolk sack; at a lower frequency there are groups of cells that look

like embryonic and adult tissues, such as muscle, cartilage and nerve (e.g. see Mostofi

& Price, 1973; Pugh, 1976).

In the serum of patients with teratomas there are found secreted products that are

characteristic of extra-embryonic foetal tissues; these include the beta subunit of

Key words: teratoma, neurons, fibronectin.

38 5. Thompson and others

human chorionic gonadotrophin (/J-HCG), alphafoetoprotein (AFP), and the am-niotic form of fibronectin. Some teratoma-derived cell lines have been shown to formthese products in xenografts or to secrete them into culture medium (reviewed byRaghavan, 1981). However, the identity of a multipotential stem cell would only befirmly established if a single cell, picked from a human testicular tumour, could begrown up to form at least some of these products or some of the characteristics ofdifferent embryonic tissues. So far, cells cloned from human testicular tumours haveshown only a limited ability to differentiate (reviewed by Andrews, Goodfellow &Damjanov, 1983; Mcllhinney, 1983; Stern, 1984). At low frequency, one clone(N2102Ep, clone 2A6) will form cells expressing /J-HCG (Damjanov & Andrews,1983); this clone was derived from the human testicular teratocarcinoma line, 2102Ep(Andrews et al. 1980, 1982). There is also a series of clones derived from the cell lineTera-2; under suitable conditions these clones have been shown to differentiate intoseveral cell forms, including neuron-like cells, which express neurofilament antigenicdeterminants and tetanus toxin receptors (Andrews, 1984; Andrews et al. 1984).These clones were isolated from tumours formed by Tera-2 in nude mice; they arecalled NTera-2 clones (Andrews, 1984; Andrews et al. 1984).

We describe the isolation of additional clones from the original Tera-2 cell line,which had not been passaged through a nude mouse (F0gh & Trempe, 1975). We havecharacterized the progressively growing small cells in cultures of these clones bystudying their surface phenotype, their secreted products, and their ability to formother cell types in culture.

MATERIALS AND METHODS

Isolation and culture of clones

The Tera-2 cell line was a gift from Professor J. F0gh (F0gh & Trempe, 1975). The cells weregrown in the alpha modification of minimal essential medium lacking nucleosides and deoxynucleo-sides (Stanners, Eliceiri & Green, 1971; Gibco Europe, Paisley, Scotland), with the addition of10% (v/v) heat-inactivated foetal calf serum (called alpha 10% FCS). Small cells with a highnuclear-to-cytoplasmic volume ratio were abundant in cultures that continued to grow progressive-ly, and cells with this form were subsequently called undifferentiated cells (Fig. 1). For routinepassage, the cells were exposed to trypsin for the minimum time to effect disaggregation (TVP;Bemstine, Hooper, Grandchamp & Ephrussi, 1973). After sucking off the TVP, the cells werebriefly incubated in EGTA/PBS, which contained 0-5mM-EGTA in PBS lacking calcium andmagnesium ions (solution A of Dulbecco & Vogt, 1954, called PBS). The cells were vigorouslypipetted from the bottom of the dish after the addition of 2-4 ml of alpha 10% FCS, spun down,and plated out at approximately 1 X 106 per 50 mm diameter culture dish containing 5 ml of alpha10 % FCS pre-equilibrated with 5-7 % (v/v) CO2 in humidified air at 37°C (dishes from Sterilin,Richmond, Surrey). The cells adhered firmly to tisssue culture surfaces that had been previouslytreated by incubation with a 0-1 % (w/v) aqueous gelatin solution for 2 h at 5 °C, and then air driedat 40°C for 24h (swine skin, type 1 gelatin, from Sigma, Poole, Dorset).

The clones were established from bulk cultures of the Tera-2 cells at the 41st passage of the line.Two days before cloning, each well of a 24-well plate was filled with 1-2 ml of alpha 10% FCS(plates from Gibco Europe). In some experiments, the surface of each well was covered with 2 X105 to 4 X 105 mitomycin-treated 10TJ cells (Reznikoff, Bertram, Brankow & Heidelberger, 1973;from M. Williams, Dunn School of Pathology, Oxford; treated as described by Martin & Evans,1975).

Near-confluent 25 cm2 Gibco Europe flasks of the cell line were refed with medium between 1 and

Differentiation of human teratoma cells 39

2h before the flasks were shaken 20 times to dislodge cells. The detached cells were pipetted intoa 90 mm diameter bacteriological grade dish on a dissecting microscope stage (Wild M5). About 20single cells were sucked into the tip of a mouth-controlled micropipette, which had been drawn toan internal tip diameter of approximately 150 [an (hard glass capillary tubing, BDH, Poole, Dorset).Single cells were blown into 50^1 drops of alpha 10% FCS, which had been placed on the surfaceof another bacteriological dish and covered with liquid paraffin (Boots Pure Drug Co., Notting-ham) . Each drop was scored for the presence of a single healthy cell using an inverted phase-contrastmicroscope (Wild M40), and such cells were transferred to individual wells of the 24-well plate usinga different micropipette for each transfer.

After 10 days of incubation, 0-5 ml of fresh medium was added to each well, and in each of thesucceeding 6 weeks half the medium was replaced with fresh medium. As each colony becameconfluent in the well, so it was sucked off the bottom with a wide-mouthed micropipette, andtransferred to a 25 cm2 flask containing 10mlofalphal0% FCS. These flasks were either untreated,covered with 10Ti mitomycin-treated feeder cells, or coated with gelatin.

The cells in these flasks were described as passage 1 cells. Thirty two of these contained somegroups of undifferentiated cells (Fig. 1); growth of these cells was usually obvious after 3 weeksincubation in flasks, but one undifferentiated cell colony did not appear until after 3 monthsincubation and it remained slow-growing. No further undifferentiated cell colonies appeared duringthe next 8 months. A variety of cell morphologies were seen in the flasks and the undifferentiatedcells continued to grow only in 25 flasks. Cells in these flasks could be passaged and their karyotypeswere scored at passages 1 and 2. In the preparation of the karyotypes, the cells were scraped fromthe dishes with a rubber policeman, briefly centrifuged, and the pellet treated with hypotonic 0'56 %(w/v) KC1 for 6 min at room temperature. They were briefly centrifuged, and the pellet was fixedin methanol/acetic acid (3:1 , v/v), and the cells were air dried onto clean dry microscope slides.The slides were 'aged' for between 3 and 10 days at room temperature, and then G-banded usinga modification of the combined ASG/trypsin method of Gallimore & Richardson (1973).

Dr Peter Andrews provided NTera-2, clone B9 (Andrews et al. 1984) and N2102Ep, clone 2A6(Andrews et al. 1982); these were tested at passages 17 and 87, respectively.

Retinoic acid treatment in monolayerTo induce differentiation we used a high concentration of retinoic acid, as recommended by

Andrews (1984). Gelatin-coated 90 mm diameter tissue culture dishes were preincubated for 1-2 hwith 15 ml of alpha 10% FCS containing 5 X 10~5M all trans retinoic acid diluted from a stock of1 X 10"1 M-retinoic acid in dimethylsulphoxide stored at —70°C (Eastman Kodak retinoic acid).This medium was called alpha RA. To obtain an even dispersal of cells across the dish, populationsof undifferentiated cells were disaggregated and between 8 X 105 and 1-5 X 10* of the cells weretaken up in the warm alpha RA medium from each dish and quickly pipetted back on to the dish,which was immediately returned to the incubator.

Aggregation and retinoic acid treatmentIn an attempt to provoke more extensive differentiation, we grew the cells in free-floating lumps.

Single undifferentiated cells adhere weakly to each other once they are in suspension, and to obtainaggregates the undifferentiated cell populations were given a brief exposure to trypsin, so that cellclumps could be blown off the bottom of the dishes after the addition of alpha 10% FCS. Theseclumps were either placed directly into 90 mm diameter bacteriological dishes, or cultured for5-16 h at high density in 50 mm diameter bacteriological dishes to promote the formation of biggeraggregates before transfer to the larger dish. Usually, retinoic acid at between 5 x 10~s M and 5 X10"7M was added to the dishes 1 day after aggregation. One week to 10 days after aggregation, theaggregates were plated out on to gelatin-coated tissue culture dishes in the presence or the absenceof retinoic acid (see Results).

TumorigenicityApproximately 1 X 107 undifferentiated cells were gently disaggregated as above, and incubated

for 5-16 h in the bottom of a 10 ml plastic centrifuge tube under 2-3 ml of alpha 10% FCS topromote the formation of a coherent lump. This was transferred as a slurry beneath the left kidney

40 S. Thompson and others

capsule of a nude mouse using a wide-bore mouth-controlled micropipette (technique of Rayner &Graham, 1982). The mice were palpated at weekly intervals to detect tumour growth, and thetumours were weighed, fixed and sectioned as described previously (Rayner & Graham, 1982; lies,Bramwell, Deussen & Graham, 1975).

Foetus samplesHuman embryonic liver and yolk sack were obtained from suction abortions. The foetal aspirates

were washed out of the collecting vessel with an equal volume of dextrose saline within 5 min of theremoval of the foetus. Within the next 2h, the material was washed free of blood by rinsing thesample with 1-2 litres of ice-cold PBS through a plastic sieve with a mesh size of approximately3 mm2. The liver and yolk sack could be easily recognized in the retained material when it had beenwashed from the sieve into a 90 mm diameter bacteriological dish and viewed under a dissectingmicroscope. The identity of the liver samples was also checked by sectioning small fragments. Forlabelling, the yolk sack was torn open and the liver was cut into cubes approximately S mm . Eachsample weighed between 0-01 and 0-02g (wet weight) and within 3-4 h of foetal aspiration each wasplaced in 1-2 ml of radioactive medium (see below). The age of the samples after fertilization rangedbetween 5 and 7 weeks as estimated from the time of the last menstrual period.

ImtnunofluorescenceIn suspension. Cells were taken from the dishes with either EGTA/PBS or with 0-125 % (w/v)

trypsin in PBS containing 0-5 mM-EDTA (EDTA/trypsin). The cells were resuspended at 1 X10 cells per ml in alpha medium with 0' 1 % (w/v) sodium azide and 5 % (v/v) FCS; 25 \i 1 sampleswere added to portions of various first-layer antibodies and incubated at 4°C for 45 min. Followinga single wash, the cells were resuspended in a 1/30 dilution of rabbit anti-mouse immunoglobulinG (IgG)-fluorescein isothyocyanate (FITC) for 45 min at 4°C. After washing, the cells were moun-ted on slides and viewed under a Zeiss IV epifluorescence condenser microscope. At least 100 cellswere counted in each sample.

Labelling of cells on coverslips. Antibodies were diluted as described under Reagents (see below)in complete phosphate-buffered saline (Dulbecco & Vogt, 1954) containing lOmM-sodium azideand either 10 % (v/v) FCS, 1 mg/ml bovine serum albumin (BSA) or 1 mg/ml gelatin. For stainingof antigens exposed on the cell surface, live cells attached to coverslips were incubated in 80^*1 offirst antibody for 30-40 min at room temperature (RT). The coverslips were washed three times bygentle immersion in PBS/azide/FCS and 80fi\ of second antibody was applied for a further30-40 min. After washing as above, the cells were fixed in 4% (w/v) paraformaldehyde in PBS(10min, RT), and the coverslips were mounted in 50% (v/v) glycerol/PBS, sealed with nailvarnish, and viewed under a Leitz microscope equipped with epi-illumination.

The intracellular neurofilaments and glial fibrillary acidic proteins were stained by fixing the cellsin paraformaldehyde as above, permeabilizing the cells in 0-1 % (w/v) Triton X-100 in PBS (RT,10 min), and then exposing them to the first antibody as described above.

Labelling for analysis on the fluorescent-activated cell sorter (FACS). Cells were released fromthe culture surface as described for cell passage. They were transferred to 10 ml plastic centrifugetubes (Sterilin), and washed in ice-cold PBS/azide with the addition of either FCS, gelatin or BSAas described above. After aspirating the supematants, cell pellets of approximately 5x10* cells weregently dispersed by tapping the tubes, and then incubated in 100 /U1 of first antibody on ice withoccasional shaking for 45 min. After two washes in 10 ml of medium, the cells were incubated for45 min on ice in 100 ji 1 of second antibody with occasional shaking. The cell suspension was dilutedto 0 5 ml and analysed on a Becton Dickinson FACS II; 100000 cells were analysed per labellingusing a 100/im diameter nozzle, and the appropriate positive and negative control antibodies wereused to set the gates.

CytophotometryCells grown on quartz coverslips were fixed in a 10% (w/v) buffered neutral formalin for 12-24 h.

This fixation stabilizes DNA during Feulgen hydrolysis and minimizes intercellular variability instaining. The cells were stained by the Feulgen reaction with hydrolysis at 22°C in 5 M-HC1 for 1 h.The cytophotometric measurements were performed in a rapid scanning microspectrophotometer


equipped with a field-limiting device (Caspersson & Lomakka, 1970; Caspersson, 1979; Caspersson& Kudynowski, 1980; Caspersson, Auer, Fallenius & Kudynowski, 1983). Imprints from humancerebellum were prepared and used as staining controls. Such nuclei contain DNA values corres-ponding to the normal diploid content (2-OC), with a variation in the majority of cells between 1 -9and 2-1C. Occasionally, cerebellar nuclei with 2-3C values were recorded and this value is taken asthe upper limit of DNA values from a diploid nucleus; all measured DNA values over Tera-2 nucleiwere expressed in C units with respect to the cerebellar controls.

In order to test if any quantitative errors were introduced when small strongly light-absorbingobjects were measured, the measurements were also performed off-peak at 610 nm, at whichwavelength the extinction was about 40 % of that obtained at the absorption peak around 546 nm.Independent of cell type, the nuclei of control cells and of Tera-2 exhibited a constant ratio ofextinction at the two wavelengths. This shows that no error of any quantitative significance wasintroduced when measuring small light-absorbing cells with compact chromatin.

Radioactive labelling and immunoprecipitationNear-confluent cell cultures were labelled for 20-26h in leucine-free MEM (Gibco Europe),

containing 0-5-5% (v/v) dialysed FCS, and between 40 and 200 jiCi/ml of L-[4,5-3H]leucine (sp.act. 131 Ci/mmol, Amersham International pic, Amersham, Bucks). In 35 mm diameter dishes,2ml of medium over 5 X 105 to 9 X 105 cells was used, and this was increased to between 3-5 and5 ml over 1 X 106 to 2 X 106 cells in 50 mm diameter dishes.

A variety of culture conditions was used to increase the chance of detecting the possible synthesisand secretion of extra-embryonic foetal products. These included aggregating monolayer culturesthat had been exposed to retinoic acid for 7 days, and culturing monolayers exposed to retinoic acidfor 4 days in alpha 2 % FCS with the addition of either 0-1 % (v/v) gelatin (swine skin, type 1 fromSigma), or 0-5 unit/ml insulin (Sigma), or 5 X 10~7M 17B-oestradiol diluted from a 1 X 10~2Mstock in dimethylsulphoxide (DMSO) (Sigma), or combinations of these substances for a further4 days before labelling.

Metabolically labelled cells and supernatants were collected and immunoprecipitated as follows.Supernatants were collected and cleared of cell debris by micro-centrifugation at 13 000 g for 10 min(volume 2ml or less) or centrifugation at 35 000 # for 15 min (greater than 2ml). Cells weresolubilized and removed from dishes by scraping them off the dish with a rubber policeman in PBScontaining 1 % (v/v) Nonidet P40 (NP40) and 0-1 mM-phenylmethylsulphonyl fluoride (PMSF)as a proteolytic inhibitor. After repeated vortex mixing over a 30-min period at 4°C, the solubilizedcellular components were removed from any remaining cellular debris by micro-centrifugation.Tissues and cell aggregates were treated as above.

The amount of radioactivity incorporated into protein was determined by precipitation withtrichloroacetic acid of small portions of supernatants and solubilized cells. All samples were thendivided into fractions and stored at — 70°C before immunoprecipitation.

Cell surface proteins were labelled by the lactoperoxidase/ I method exactly as previouslydescribed (Stern et al. 1984).

Immunoprecipitation was carried out by incubating 100 to 500/il portions of supernatants and100^t 1 portions of solubilized cells with 10/zl of the appropriate antibody for 30min at roomtemperature. To each tube was added 50/il of a 50% (w/v) suspension of protein A-Sepharose(Pharmacia) in 1 % (v/v) NP40 in PBS, and the samples were incubated again for 30 min at RT.Unbound material was then washed away from the Sepharose beads by five washes at 4°C undergravity or brief centrifugation in 2-5 ml of 1 % (v/v) NP40 in 50mM-Tris HC1 (pH 7-4), contain-ing 0-15 M-NaCl, 5mM-EDTA and 0-02% (w/v) NaN3. Finally, the antigen-antibody complexeswere removed from the beads by the addition of 50/il of 2X concentrated sodium dodecyl sul-phate/polyacrylamide gel electrophoresis sample buffer lacking 2-mercaptoethanol (2-ME), and thesupernatants were harvested after micro-centrifugation. The samples were boiled after the additionof 5 % (v/v) 2-ME and subjected to electrophoresis in 5'5 %, 8-0% and 13 % (w/v) polyacrylamidegels, using the method of Laemmli (1970), as modified by Thompson & Maddy (1982). Molecularweight standards were red blood cell membrane-protein bands 1,2,4-1,4-2, 5,6 and 7 with molecularweights of 240, 220, 78, 72, 43, 35 and 29 (X103), respectively, and a 14C-labelled methylated proteinmixture containing myosin (200 X \C^MT), phosphorylase 6 (92-5 X KfiM^), bovine serum albumin(BSA, 69 X \(PM,), ovalbumin (46 X UfiM,), carbonic anhydrase (30 X l(PMT), and lysozyme


(14-3 X 103iVfr). In comparisons between undifferentiated and differentiated cell labelling,precipitation analysis was carried out by applying the antibody to samples that contained equaltrichloroacetic acid-precipitable counts. The samples were counted at an efficiency of approximately40% in a LKB 1215 Beta counter.

Preflashed Fuji X-ray film was used for autoradiography with Dupont Cronex Lighting Plusscreens (Laskey & Mills, 1977), and fluorography was conducted with scintillant-impregnated gels;both procedures conducted at -70°C (Bonner & Laskey, 1974; Laskey & Mills, 1975).

ReagentsThe specificities and originators of the reagents are described in Table 1. We thank the following

for gifts of reagents (dilutions used): Dr A. Williams, Dunn School of Pathology, Oxford, forW6/32 (culture supernatant) and W3/25 (culture supernatant); Dr J. Fabre, Queen VictoriaHospital, East Grinstead, Sussex, for F10.44.2 (1/100 ascites) and F15.42.1 (1/50 ascites); Dr D.Solter, Wistar Institute, Philadelphia, for anti-SSEA-1 (1/100 culture supernatant) and anti-SSEA-3 (culture supernatant); Dr M. Schachner, Department of Neurobiology, University ofHeidelberg, 69 Heidelberg, Im Neveheimer Feld 504, FDR, for 04 (1/1000 ascites); Professor A.McMichael, John Radcliffe Hospital, Oxford, for PA 2.6 (1/100 ascites) and MHM 5 (1/100ascites); Dr R.O. Thomson, Wellcome Laboratories, Beckenham, Kent, for tetanus toxin (10mg/ml), affinity-purified horse anti-tetanus toxin antibody (1 /50), and fluorescein rabbit anti-horse(1/20); DrJ . Kemshead, ICRF Laboratories, Institute of Child Health, Guilford Street, LondonWC1, for U.13A (1/500 ascites), 308 (1/50 direct FITC conjugate) and MIN 1 (1/100 ascites);Dr B. H. Anderton, Department of Biochemistry, St George's Hospital Medical School, LondonSW 17, for BF10 (1/1000 ascites) and RT97 (1/200 ascites).

The other antibodies were prepared in the laboratory and used at the following dilutions: F12A2B5 (1/100 ascites), 2-3 F9 (1/100 ascites), anti-GFAP (1/50 serum prepared as described byPruss, 1979).

Other antibodies were obtained from the following: rabbit anti-human fibronectin (Dr D. Tur-ner), and rabbit anti-apoprotein Al, anti-albumin, anti-transferrin, anti-AFP, anti-prealbumin,anti-/3-HCG, from Boehringer; mouse anti-/32-microglobulin directly labelled monoclonal, fromBecton Dickinson. Second-layer antibodies were rabbit anti-mouse IgG-FITC (Miles, 1/60 to1/100), and rhodamine-labelled sheep anti-mouse and anti-rabbit IgG (1/40).

RESULTS

The results are divided into four sections. First, the growth and morphologicaldifferentiation of the clones are described, and then we record the changes in surfacephenotype and secreted products that accompany their differentiation in retinoic acid.Finally, we characterize the development of a neuron-like phenotype that forms afteraggregation and treatment with retinoic acid.

Clonal growth, karyotypes and morphological differentiation

In the cloning procedure, it was apparent that isolated single cells of Tera-2required feeder cells if they were to form colonies: none of the single cells formedvisible colonies in alpha 10% FCS alone (« = 75), while 41% grew into visiblecolonies during the 3 weeks after the single cells had been plated on 10T^ feeder cells(n = 113). However, none of the clones required feeder cells once they were suf-ficiently numerous to be passaged at high cell density.

A number of different cell morphologies were seen at the first passage of the clones.Cells with long branching processes were found in groups in 7/25 of the clones. Weselected clones 5, 12 and 13 for detailed study because they could be maintained with


Fig. 1. Tera-2 clone 13 at passage 3. The piled up and monolayer forms of undifferen-tiated cells occupy most of the photograph. In the monolayer at the top right, the highnuclear-to-volume ratio is clear. At the bottom left are several differentiated cells; thesehad formed spontaneously but they resemble the cell shapes formed in response to retinoicacid. Bar. SO/im.

a relatively homogeneous appearance. The form of the majority of cells in untreatedpopulations of these clones is described as undifferentiated (Fig. 1).

The karyotypes of clones 5, 12 and 13 were all abnormal and the cells in each clonehad a range of mitotic chromosome counts within two passages of cloning: clone 5 hada modal chromosome number of 58 (range 57-59, n = 10); clone 12 had a modalchromosome number of 63 (range 62-65, n = 8); clone 13 had a modal chromosomenumber of 60 (range 57-63, n = 100). These chromosome numbers are similar tothose found in uncloned Tera-2 cells and in the NTera-2 clones (Andrews et al. 1984).

The G-banded karyotype of Tera-2 clone 13 was analysed in detail and its featuresshowed some similarity to those previously described for uncloned Tera-2 and theNTera-2 clones (Andrews et al 1984). There were the following normal-lookingchromosomes (Fig. 2): one copy of chromosomes Y and 1; two copies of chromosomes2, 4, 5, 6, 8, 10, 12, 13, 14, 15, 17, 18, 19, 20 and 22; and three copies of chromosomes3, 7, 9, 11 and 21. The presence of rearranged chromosomes resulted in furtherrecognizable partial trisomies and tetrasomies. For example, there were threeadditional copies of chromosome 1, each with the short arm deleted in different

5. Thompson and others

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places; the metacentric chromosome derived from the short arm of chromosome 1translocated to the short arm of chromosome 2 (Andrews et al. 1984), and achromosome 16 with a deleted short arm. The single X chromosome was abnormal,with a wide positive band in the long arm at q25. Neither the previously reportednumerous chromosome 12 involvements, the isochromosome of the long arm of 17,nor the deleted chromosome 22 were seen (Andrews et al. 1984). A number ofcharacteristic marker chromosomes was observed but we could not confidently deter-mine which chromosomes had contributed to these extra elements. To reduce thepossibility that changes in the modal chromosome number might alter the behaviourof the clones during the course of these experiments, we made our observations oncells that had been grown for less than 16 passages after cloning.

Clones 12 and 13 were shown to form tumours at 2-3 months after inoculation ofthe cells into nude mice. The tumour formed by clone 12 cells weighed 0-032g, andthe two tumours from clone 13 cells weighed 0-014 and 0-055 g. On sectioning thesewere found to contain groups of cells with the appearance of carcinoma, loose connec-tive tissue and cuboidal epithelia around cavities, and there were long processes deepin the tumour that had the appearance and staining properties of nerve axons (Figs3-6). There were also cells with a variety of other morphologies. These tumoursresembled those formed by uncloned Tera-2 in nude mice (Jewett, 1978). Unlike thetumours formed by the NTera-2 clones, glandular epithelia were not found, and thetumours also lacked the cells described by histopathologists as 'embryonal carcinomacells arranged in papillary formation', which are found in primary teratomas.

After exposure to retinoic acid in monolayer culture, the cell numbers in the dishesincreased approximately fivefold in the next 7-10 days. Measurements of amounts ofnuclear DNA in the undifferentiated and treated populations showed that within 8days the cells transit from a rapidly dividing state, with DNA amounts ranging from3C to 8C, to a population in which the majority of the cells are arrested in the G\ phaseof the cell cycle displaying 3C nuclear DNA amounts (Figs 7—10). In many experi-ments, the retinoic acid was withdrawn at this time, and the cell number did notincrease further during the following 2 months.

A variety of cell morphologies was seen in the treated cultures. The undifferen-tiated cells seem to have disappeared from these cultures because their characteristicmorphology was not apparent after an extensive search of several hundred retinoic-acid-treated cultures. Further, when the cells were cultured on for several months inthe absence of retinoic acid, then in four separate experiments with both clone 12 andclone 13 cells, the undifferentiated morphology did not recur.

Changes in surface phenotype on differentiation in monolayer culture

We characterized the phenotype of undifferentiated cell populations and of the

Fig. 2. G-banded karyotype of Tera-2, clone 13:61, X (abnormal) Y, - 1 , +del 1, +del1, +del 1, +t(l; 2), +3, +7, +9, +11, +del 16, +21, +mar, +mar, +mar, +mar, +mar,+ mar (?12h). Although there were variations in the marker chromosomes, the otherfeatures described in this legend were apparent in each of the 10 karyotypes analysed indetail.


ms^if

Fig8 3-6


differentiated cells formed in monolayer cultures in response to retinoic acid. Theantigenic phenotype of the undifferentiated cells was distinct from that of the NTera-2 clones, and there were striking changes in the expression of HLA-A,B,C commondeterminants during this differentiation.

The expression of HLA-A,B,C common antigens and ^2-microglobulin wasstudied with monoclonal antibodies to these determinants (W6/32, PA 2-6, anti-jSz).The undifferentiated cell populations rarely reacted with these monoclonal antibodies(Table 2) and a similar result was obtained with MHM5, another monoclonal toHLA-A,B,C common determinants (results not shown). In those experiments inwhich more than 3 % of the undifferentiated cell populations appeared to react withthese antibodies, an independent observer had previously recorded that the popula-tions contained morphologically distinct flat cells. There is therefore a close associa-tion between the undifferentiated appearance and the lack of reactivity with theseantibodies. The majority of these observations were made by eye, but the rare reactiv-ity of undifferentiated cell populations was also confirmed on FACS. It was found that2-5 % more cells reacted with W6/32 than with an irrelevant antibody (W3/25) in asample that was scored as 3 % reactive with W6/32 by eye.

After exposure to retinoic acid for a week, there was a considerable increase in theproportion of cells that reacted with the monoclonal antibodies W6/32, PA 2.6, andthe anti-/S2-microglobulin antibody (Table 2). This observation was confirmed on theFACS using the W6/32 antibody reaction to clone 12. This new phenotype was stablefollowing withdrawal of retinoic acid after treatment for 1 week; for example, 1 monthafter withdrawal, the differentiated clone 13 cells exhibited 97% reactivity withW6/32, 100% reactivity with PA 2.6, and 100% reactivity with the anti-/S2-micro-globulin antibody. In another experiment, 4 months after the withdrawal of retinoicacid, 55% of clone 12 differentiated cells reacted with W6/32. The increase inreactivity with W6/32, PA 2.6, and anti-/J2-microglobulin antibodies was progressiveduring continuous exposure to retinoic acid: for instance, in one particular experi-ment, the proportion of clone 12 cells that reacted with W6/32 increased progressivelyto give 1-3 % reactivity at 2 days, 7 % at 4 days, 26 % at 6 days and 53 % at 9 days.The reactivity of the cells with PA 2.6 and anti-/?2-microglobulin antibodies increasedin parallel with W6/32 reactivity in this experiment.

Figs 3-6. Histology of tumours formed by Tera-2 cells grown under the kidney capsuleof nude mice.

Fig. 3. Tera-2, clone 13. The tumour principally consists of nests of carcinoma cells inloose connective tissue. Apparently normal mouse kidney tubules occupy the bottom halfof the figure. Bar, 50 (im.

Fig. 4. Tera-2, clone 13. A nest of carcinoma cells with a high mitotic index besidekidney tubules along the right of the picture. Bar, 20 |im.

Fig. 5. Tubule within a Tera-2, clone 12 tumour. The simple epithelium is surroundedby connective tissue. Bar, 20 fim.

Fig. 6. Axon-like process within a Tera-2, clone 12 tumour. It is impossible to excludethe possibility that the axon is derived from the surrounding host kidney but it was founddeep in the tumour. Holmes' silver stain. Bar, 20|tm.

cl12

5. Thompson and others

cl13

10

cl13+ RA

2C 4C 8C 10C 6C 8C 10C

DNA content (relative units)

Figs 7-10. Distribution of nuclear DNA content (Feulgen staining material) in undif-ferentiated cells and differentiated cells, formed after 8 days retinoic acid (RA) treatment.Each histogram is based on the cytophotometric analysis of approximately 100 cells. TheDNA values are expressed in relation to the mean staining values of the control cerebellumcells: these were given the arbitrary value of 2C (denoting the diploid DNA content), anda broken line on each figure represents the upper limit of the staining control (2'1C).

Fig. 7. Clone 12, undifferentiated.Fig. 8. Clone 12, differentiated.Fig. 9. Clone 13, undifferentiated.Fig. 10. Clone 13, differentiated.

The monoclonal antibody W6/32 was used to immunoprecipitate leucine-labelledmaterial from the differentiated cells and it specifically precipitated a 43 X 1037Wr

molecule, which is slightly smaller than the expected size of HLA-A,B,C polypep-tides.

The majority of the undifferentiated cells of the NTera-2 clones were known to


Table 1. Reactivities of reagents

Monoclonal antibodyor reagent(original reference) Major determinant or molecule bearing determinant (reference)

W6/32 (1)

PA 2.6 (6)MHM-5 (8)Anti-ft-microglobulin

(9)SSEA-3 (10)

SSEA-1 (13)

F10.44.2(17)

F15.42.1 (20)RT97(21)BF10(21)Tetanus toxin

F12.A2B5 (28)

GFAP (30)

U13A(31)

S.1H11 (33)MIN 1 (36)04 (39)

2.3 F9 (40)

HLA-A,B,C (44*) in association with human or mouse ft-microglobulin(12*). Weak reaction with HLA alone, none with /32-microglobulinalone (2,3,4,5). Structural gene on chromosome 6

HLA-A,B,C (44#) in association with human /32-microglobulin (12*) (7)HLA-A,B,C (44*) in association with human /32-microglobulin (12*) (8)/32-microglobulin (12*) alone or in association with HLA-A,B,C.

Structural gene on chromosome 15 (7)3GalNAc/31—»3Galal—>4Gal, on glycoproteins and glycolipids of an

human teratoma line (2102Ep); on preimplantation mouse embryosup to the 8-cell stage (10,11,12)

Gal/51—+4(Fucal—»3)GlcNAc, principally found on glycolipids; onpreimplantation mouse embryos from morula stage onwards(14,15,16)

87-89 X 103jWr sialoglycoproteins on human brain and bloodmononulear cells. Structural locus or expression control onchromosome 11 (17,18,19)

23-5 X 1037tfr, human Thy-1 (20)210 X \02MT neurofilament on human brain (21)155 X 103Mr neurofilament on human brain (21)Reacts with gangliosides with decreasing affinity, GTlb,GDlb,GDla.

Reacts with neurons, astrocytes, pancreatic cells, thyroid cells andpresent on 10th day mouse brain (22,23,24,25,26,27)

Highest affinity for GQlc gangliosides. Reacts with neurons, someastrocytes, some pre-oligodendrocytes and 'APUD' cells (25,28,29)

Glial fibrillary acidic protein. Restricted to glial cells (30)On human foetal muscle cultures, reacting with myoblasts, myotubes

immature myofibres and adult regenerating myofibres. On neurons infoetal brain cultures (32,33,34,35)

Distribution similar to U13AAstrocytes of human brain cultures and granulocytes (36,37)Mouse oligodendrocytes and galactocerebroside positive cells on human

brain cultures (35,38,39)Fibronectin (40)

The origin of the reagents and the distribution of their reactivities is described in these references.(1) Williams et al. (1977); (2) Barnstable et al. (1978); (3) Parham et al. (1979); (4) Trowsdalee/al. (1980); (5) Goodfellowet al. (1976); (6) Parham & Bodmer (1978); (7) Brodskyefa/. (1979);(8) Hildreth (1982); (9) Becton Dickinson; (10) Shevinsky et al. (1982); (11) Kannagi et al.(1983a); (12) Kannagi et al. (19836); (13) Solter & Knowles (1978); (14) Gooietal. (1981); (15)Hakomorie* al. (1981); (16) Kannagi et al. (1982); (17) Dalchau et al. (1980); (18) Goodfellowet al. (19S2a,b); (19) McKenzieet al. (1982); (20) McKenzie & Fabre (1981); (21) Andertonet al.(1982); (22) Van Heynigen (1963); (23) Lebdeen&Mellanby (1977); (24) Rogers &Snyder (1981);(25) Eisenbarth <?* al. (1982); (26) Raff etal. (1983); (27) Koulakoff et al. (1983); (28) Eisenbarthet al. (1979); (29) Kasai & Yu (1983); (30) Pruss (1979); (31) J. T. Kemshead; (32) Walsh et al.(1983); (33) Walsh et al. (1981); (34) Walsh & Ritter (1981); (35) Hurko & Walsh (1983); (36) G.Dickson, personal communication; (37) Kemshead et al. (1981); (38) Dickson et al. (1983); (39)Somner & Schachner (1981); (40) Walsh et al. (1981).

•Molecular weights are given in parenthesis (Xl0~3).


Table 2. Reactivity of undifferentiated and differentiated cells

Reagent

W6/32

PA2.6

Anti-ft

SSEA-3

SSEA-1

F10.44.2

F15.42.1

BF10

Tetanustoxin

F12A2B5

Anti-GFAP

U13A

Percentage of cells reactive: mean,

Undifferentiated populations

Uncloned

0(0)

nd

nd

0(0)4

(4)

80(80)

95(95)

nd

nd

nd

nd

nd

Clone 5

3(0-5,2,7)

nd

3(3)0

(0,0)

31(27,28,38)

92(89,95)

97(95,100)

0(0)1-1

(1-1)

0-75(0-75)

0(0)nd

Clone 12

2-2(0,0,2,3,6)

3-2(3-2)

4-5(4-5)

0(0,0)

43(20,67)

85-5(76,95)

81(67,95)

0(0)1

(0,2)0

(0)0

(0)8

(8)

Clone 13

0 1(0,0,0,0,0,0,0,0,0,9)

0(0)0

(0)0

(0,0)

49(20,22,42,51,60,100)

66(3,60,81,

89,97)

94-6(86,94,95,

98,100)

0(0)6-4

(6-4)

2-8(2-8)

0(0)nd

(individual observations)

Differentiated populations

rClone 5

18(18)

nd

nd

0(0,0)

36(36)

35(35)

39(39)

nd

nd

nd

0(0)nd

Clone 12

40-6(7,46,54,54)

56-5(40,73)

54-4(54-4)

0(0,0)

33(27,39)

53-5(20,87)

76-5(57,96)

3(3)17

(17)

7(7)0

(0)43

(43)

Clone 13

20-8(1,19,24,

25,35)

38(38)

6(6)0

(0,0)

55(0,64,100)

69(67,71)

34(34)

nd

nd

nd

0(0)nd

The reactivities of the cells were not always tested on the same day with the same reagents;because these are not matched comparisons we give the individual observations made on differentdays with different cell populations. This establishes the reproducibility of the results. Observationson cells in suspension are recorded in the top half of the Table. Studies on cells attached to coverslipsare in the bottom half of the Table.

nd, not determined.

react with W6/32 (Andrews et al. 1984). We therefore compared the reactivity of cellsfrom NTera-2 clone B9, Tera-2 clones 5 and 12, and the cloned 2102Ep cells, whichwere also known to react with W6/32 (Table 3). It is clear that the expression of HLA-A,B,C determinants distinguishes the two sets of clones that were independentlyderived from Tera-2.


Table 3. Comparison ofNTera-2 clones, Tera-2 clones 5 and 12 and2102Ep

W6/32

SSEA-3

SSEA-1

F10.44.2

F15.42.1

Comparisonsmerit using the

Undifferentiated cell populationK

Clone 5

<1

0

27

>95

>95

Clone 12

0

0

67

>95

>95

between the reactivitysame reagents for each,

N.Tera-2 2102Ep cloneclone B9 2A6

24

87

27

>95

>95

53

>9S

2

>95

>95

Differenti:popula

A(

Clone 5

18

0

36

35

39

ited celltions

Clone 12

7

0

27

20

57

of these cells with various reagents was made in one experi-

In the same experiment, Tera-2 clones 5 and 12 were further distinguished fromthe NTera-2 clone by the lack of reactivity with the anti-SSEA-3 monoclonal anti-body. This monoclonal antibody recognizes a sugar sequence that is expressed in theearliest stages of preimplantation mouse development (Table 1), and it has beenproposed that it identifies human teratoma cells at an analogous stage of development;it reacts with the NTera-2 cells and the 2102Ep cells (Shevinsky, Knowles, Damjanov& Solter, 1982; Andrews et al. 1984). The SSEA-3 determinants are clearly absentfrom the morphologically similar Tera-2 clones 5, 12 and 13, and they do not appearon differentiation (Tables 2 and 3).

The monoclonal antibody against SSEA-1 recognizes determinants that appear ata later stage of preimplantation mouse development than those recognized by theantibody against SSEA-3 (Table 1; Solter & Knowles, 1978). This reagent reactedwith between a third and an half of the" cells from clones 5, 12 and 13 in both theundifferentiated and differentiated populations (Table 2 and 3). About a quarter ofNTera-2 clone B9 cells reacted, but only a few 2102Ep cells reacted.

Most human teratoma-derived lines with an undifferentiated appearance expressThy-1 (Andrews et al. 1984), and the undifferentiated cells of clones 5, 12 and 13behaved similarly when tested with the monoclonal antibody F15.42.1; on dif-ferentiation there was a consistent decrease in the proportion of cells that expressedthis determinant (Table 2 and 3).

The monoclonal antibody F10.44.2 recognizes an approximately 90 X 103Mr

sialoglycoprotein that is present on human brain and white blood cells. This reactivitywas also present on the cells of Tera-2 clones 5, 12 and 13, as well as on NTera-2 cloneB9 and 2102Ep cells (Tables 2 and 3). The undifferentiated cells of Tera-2 clones 5,12 and 13 were surface-labelled with lactoperoxidase/12SI and the detergent-solubilized labelled proteins were precipitated with this antibody'. The molecule thatwas immunoprecipitated ran as a broad band, as would be expected of a glycoprotein,with an apparent molecular weight of 85 to 95 X 103Mr. This immunoprecipitate is


shown for clone 13 cells in Fig. 11. Although the proportion of the cells that react withF10.44.2 decreases on differentiation, there is no change in the molecular weight ofthis protein (data not shown). Fig. 11 also illustrates other general changes in cellsurface phenotype that occur after retinoic acid treatment. For example, proteins ofmolecular weights 260, 200, 75, 52, 50, 48 X l^Mr seem to disappear and aprominent band appears at a molecular weight of 67 X lCPM,-.

Changes in synthesized proteins and secreted products on differentiation

The pattern of protein synthesis changed on retinoic acid treatment, and thischange was obvious both in cell lysates and in the secreted products of the cells (data

1 2 3

240220

7872

43

35

29

Fig. 11. Cell surface labelled components of undifferentiated clone 13 (lane 1), anddifferentiated clone 13 (lane 2). The material immunoprecipitated by the monoclonalantibody F10.44.2 from the undifferentiated cell sample is shown in lane 3. The sampleswere all run on the same 8 % polyacrylamide gel, but a longer exposure was required fordetection of the immunoprecipitated molecule. MT values are given (X 1(T3) on the right.


not shown). The synthesis of fibronectin was examined, since this protein is oftenexpressed as human teratoma cells differentiate (Andrews, 1982). The anti-fibronectin antibody was used to precipitate material from the culture medium over

1

f

* * > • •

-200

-100-92-5

Fig. 12. Labelled cell culture medium treated with anti-fibronectin antibody. Undifferen-tiated clone 13 (lane 1), differentiated clone 13 (lane 2), foetal yolk sack (lane 3), foetalliver (lane 4), undifferentiated clone 12 (lane 5), differentiated clone 12 (lane 6) anddifferentiated clone 12, after incubation with gelatin, insulin and /3-oestradiol (lane 7). Fmarks the position of fibronectin. Other radioactive bands were specifically and repeatedlyimmunoprecipitated by this antibody. Immunoprecipitation was carried out on sampleswith 480000 c.p.m. of trichloroacetic acid-precipitable material, except for the yolk sacksample, which contained 100000 c.p.m. The total immunoprecipitate was applied to thegel, except for differentiated clone 12 samples, where only 40 % of the immunoprecipitate(lane 6), or 20% of the immunoprecipitate (lane 7) was added to the gel. Fluorograph ofa 5 5 % polyacrylamide gel.

54 iS. Thompson and others

undifferentiated and differentiated cells; the electrophoretic analysis of theprecipitated material for Tera-2 clones 12 and 13 is presented in Fig. 12. The undif-ferentiated cells of Tera-2 clones 5, 12 and 13 secreted fibronectin, and this had anapproximate molecular weight of 240 X 103. All the differentiated cultures apparentlysecreted proportionately more of this protein compared to the undifferentiated cellcultures. On differentiation, the apparent molecular weight of the secreted fibronec-tin decreased by about 5 X \{9Mr (Fig. 12).

It was possible that a multipotential stem cell from a teratoma might be induced tosecrete those products that are characteristic of the extra-embryonic membranes of thehuman foetus (see Discussion).

However, we were unable to detect the synthesis and secretion of /3-HCG, AFP,prealbumin, albumin, apoprotein Al or transferrin from labelled undifferentiated anddifferentiated cells or from the culture medium over these cells. In contrast, all theseproducts except HCG were secreted into the medium by the human foetal liver andyolk sack. We were able to immunoprecipitate specifically AFP, albumin, apoproteinAl and transferrin from these tissues and from the culture medium conditioned bythese tissues, even when the samples contained down to 30-fold less counts in totaltrichloroacetic acid-precipitable material.

Appearance of cells vnth a neuron-like phenotype

The undifferentiated and differentiated cells in monolayers rarely reacted withreagents that react with human neurons (Table 2). A few cells with long processeswere seen amongst the variety of cell morphologies formed in response to retinoicacid. The proportion of cells that reacted with reagents that mark neurons increasedon retinoic acid treatment; these reagents included tetanus toxin, F12 A2B5, U13Aand5.1Hll (Table 2).

Cells with long branching processes were obvious in cultures that had been exposedto retinoic acid as aggregates, and then grown on, after a week's treatment in eitherthe presence or absence of retinoic acid; continued treatment with retinoic acid hadlittle effect on the results (Figs 13—18). All the cells with long processes in suchcultures of clone 12 reacted with the anti-neurofilament monoclonal antibody RT97(where number of cells observed (n = 46), with the anti-specific ganglioside reagenttetanus toxin (n = 32), with the anti-GQlc ganglioside monoclonal antibody F12A2B5 (n = 14), with the anti-Thy-1 monoclonal antibody F15.42.1, and with twomonoclonal antibodies that are characteristic of excitable cells, namely U13A (n = 13)and 5.1H11 (n = 13) (Figs 13-18). None of the cells reacted with the monoclonalantibodies MIN 1 (n = 42), 04 (n = 20), or with the glial marker anti-GFAP(n = 100). Similarly, all cells with long processes in previously aggregated and treatedcultures of clones 5 and 13 reacted with the anti-neurofilament antibody BF10. Ineach experiment, the proportion of cells that reacted with the anti-neurofilamentantibodies varied between 1 and 3 %.

Three of the clones that spontaneously formed cells with branched processes at firstpassage were tested for their ability to express neuron markers when they differen-tiated at high density in the absence of retinoic acid. Clones 4, 25 and 29 displayed


15

Figs 13-18. Tera-2 expression of antigens that are characteristic of the nervous system.Tera-2 clone 12 cells were aggregated in the presence of retinoic acid, and the aggregatesgrown up for a further 4 weeks (see Materials and Methods). They were stained withtetanus toxin (Figs 13, 14), with F12 A2B5 monoclonal antibody (Figs 15, 16), and withthe monoclonal antibody to neurofilaments, BF10 (Figs 17, 18). Figs 13, 15 and 17,photographed with phase optics. Figs 14, 16 and 18 are the paired fields photographedunder fluorescent illumination. Bars: Fig. 13, 100 fim\ Figs 15 and 17, 50/an.

cells with processes that stained with the anti-neurofilament monoclonal antibodyBF10, and when tested these cells also reacted with tetanus toxin and F12 A2B5.


DISCUSSION

Recognition of the undifferentiated cell

The morphology of the rapidly growing small cells in our cultures resembles thatdescribed for the NTera-2 clones and that observed in a variety of other teratoma-derived cell lines (e.g. see Andrews et al. 1980). Fig. 1 shows that these Tera-2 cellsusually have a more flattened appearance than NTera-2 cells (e.g. see Andrews et al.1984, fig. 1A), although they retain the high nuclear : cytoplasm profile ratio of thegrowing NTera-2 cells. We have called it an undifferentiated cell (Fig. 1). The mainreason for believing that single undifferentiated cells can develop into cells with thevariety of morphologies seen in these experiments, is that most of the cells in the Tera-2 cultures that were cloned looked undifferentiated; 41 % of single cells picked fromthese cultures to feeder cells subsequently grew on, to form passage 1 colonies inwhich a wide range of morphologies could be seen, including cells with long processes.There are several reasons for believing that they are the only progressively growingcells in cultures of Tera-2 clones 5, 12 and 13. First, cells with this morphology wereabundant in all passage 1 bottles in which cells continued to multiply, and when theywere absent or disappeared from passage 1 bottles, then the remaining cells did notgrow progressively over an eight-month period. Second, the undifferentiated cellfeatures disappeared on retinoic acid treatment, and the treated populations did notgrow progressively after retinoic acid had been withdrawn for many months. Suchcultures did not grow progressively, even when the cells were sub-confluent.

We have studied clones that were relatively easy to grow as cell populations in whichthe majority of cells looked undifferentiated. Nevertheless, it was common to observethat about 5 % of the cells had a more flattened profile. The origin of this cell type isnot known. It is likely that the appearance of such cells in undifferentiated culturesindicates that a small proportion of the cells have spontaneously started on the changesthat lead towards the phenotype of the retinoic acid-treated cells, whose form theyresemble. Similar 'spontaneous' differentiation has also been observed in other studieswith Tera-2 clone 13 cells (Engstrom, Heath & Rees, unpublished data). It is alsopossible that such cells could have been produced by chromosome changes in thepopulation because there was considerable variation in mitotic cell chromosome num-bers in individual clones within two passages of cloning. We may not have eliminatedthis possibility by restricting these observations to the first 15 passages after cloning.

Cell surface phenotypic changes

Despite the common ability of both these Tera-2 clones and the NTera-2 clones toform neuron-like cells, there were considerable differences between the phenotype ofthe small progressively growing cells of these two sets of clones. These differenceshave been confirmed by the exchange of cells and reagents with Dr Peter Andrews.

HLA-A,B,C and $2-microglobulin. It is agreed that uncloned Tera-2 cells have few,if any, HLA-A,B,C determinants on their surface that are available for reaction withantibodies against these determinants; these determinants have been searched for


with monoclonal antibodies, human alloantisera, and rabbit antisera to purified HLA(Andrews, Bronson, Wiles & Goodfellow, 1981; Avner, Bono, Berger & Fellous,1981; Mcllhinney, 1981). The uncloned Tera-2 cells used in this work rarely reactedwith W6/32 (see Table 2). Similarly /32-microglobulin appears to be rare or absentfrom the surface of the uncloned cells; its availability for reaction with antibodies hasbeen tested with xenoantisera and monoclonal antibodies (Holden et al. 1977; An-drews et al. 1981; Avner et al. 1981; Andrews, 1983).

The three monoclonal antibodies that we have used to detect the HLA-A,B,Ccommon determinants on the undifferentiated cells of these Tera-2 clones, wouldreact avidly with these determinants only in the presence of /32-microglobulin (Par-ham, Barnstable & Bodmer, 1979; Trowsdale, Travers, Bodmer & Pattillo, 1980).Since /32-microglobulin determinants are rare or absent from the undifferentiatedcells, we are unable to exclude the possibility that the HLA-A,B,C polypeptides existas free glycoprotein. It is also possible that these determinants are not inserted intothe cell membrane, and are therefore not available for reaction with antibodies in thesecell surface assays.

We interpret the low proportion of cells in the undifferentiated cell populations thatreact with W6/32 and the anti-/S2-microglobulin antibody, as either the tail of adistribution of a population of very weakly positive cells (Andrews et al. 1981), or asevidence that some of the cells have started to diverge from the undifferentiated cellphenotype. We favour the latter interpretation because W6/32 reacting cells wereseen only when the undifferentiated cell population had been scored by an indepen-dent observer as containing 5 % or more flattened cells, and because these reactingcells became more abundant as the proportion of flattened cells increased after retinoicacid treatment.

Tera-2 clones 5, 12 and 13 resemble mouse embryonal carcinoma cells in the lackof expression of major histocompatibility complex (MHC) products (reviewed byStern, 1983). Amongst the other human teratoma-derived lines only SuSa and LICRLON HT53 rarely react with antibodies to HLA-A,B,C determinants; the latter cellline forms AFP, when it is grown as a tumour (Hogan, Fellous, Avner & Jacob, 1977;Mcllhinney & Patel, 1983).

Sugar specificities. Few cells in uncloned Tera-2 populations react with themonoclonal antibody against SSEA-3 (Shevinsky, Knowles, Damjanov & Solter,1982; Andrews et al. 1984; observations, this paper). It is therefore not surprisingthat Tera-2 clones 5, 12 and 13 also lack this sugar determinant and the overlappingepitope recognized by the antibody against SSEA-4 (Peter Andrews, personal com-munication). Although the antibody against SSEA-3 reacts with some 'embryonalcarcinoma' cells in primary teratomas (Damjanov et al. 1982), the phenotype of theseclones demonstrates that tumorigenic bipotential cells that do not react with theantibody against SSEA-3 can be derived from human teratomas.

Uncloned Tera-2 cells show variable levels of reaction with the antibody againstSSEA-1, and the stem cell populations of NTera-2 clones rarely react (Andrews et al.1980, 1984; Andrews, 1984). Since this monoclonal antibody does not react withembryonal carcinoma in primary solid human tumours, it is thought to mark more

CEL72


differentiated cells, including the differentiated cells produced by retinoic acid treat-ment of NTera-2 (Damjanov et al. 1982; Andrews et at. 1984; Andrews, 1984). Asubstantial proportion of both the undifferentiated and differentiated populations ofthe clones 5, 12 and 13 reacted with this antibody, which also reacts with the em-bryonal carcinoma cells of mouse teratoma (Solter & Knowles, 1978).

Uncloned Tera-2 cells also express a number of other sugar specificities in commonwith mouse embryonal carcinoma. The monoclonal antibody 2H9 reacts with both,as does the lectin peanut agglutinin (Bono et al. 1981; Stern et al. 1984). Further,antibodies raised against mouse embryonal carcinoma cross-react with Tera-2 cells(Ostrand-Rosenberg, Edidin & Jewett, 1977).

Extraembryonic secreted proteins

The majority of primary human teratomas contain some cells that react withantibodies against /J-HCG and other cells that react with anti-AFP antibodies;frequently the serum of such patients contains elevated levels of these glycoproteins(papers in Norgaard-Pedersen, 1978; Anderson, Jones & Milford Ward, 1981; New-lands & Reynolds, 1983). A fully multipotential stem cell from a teratoma would beexpected to be able to develop into cell types that synthesize both these products.Further, the abundance of cells in primary teratomas that resemble cells in the yolksack (e.g. see Beilby, Home, Milne & Parkinson, 1979), suggests that, in addition toAFP, the other secreted serum proteins of the yolk sack would be produced by thedifferentiated derivatives of a multipotential cell; these serum proteins includeprealbumin, albumin, transferrin and apoprotein Al (Gitlin & Perricelli, 1970; Git-lin, Perricelli & Gitlin, 1972; Shi et al. 1984).

We have been unable to detect the synthesis of any of these products under a varietyof culture conditions. This is unlikely to be due to technical difficulties because theseproducts could be readily detected in cultures of human yolk sack and foetal liver thatcontained down to 30-fold less total trichloroacetic acid-precipitable counts. Similar-ly, it has been impossible to detect the synthesis of HCG or AFP in cultures of theNTera-2 clones either before or after retinoic acid treatment (Andrews et al. 1984).

The failure to induce the synthesis of these products may either indicate that thecells are not fully multipotential stem cells, or show that it is difficult to reproduce theunknown conditions that lead to the synthesis of these products in a solid tumour; theculture conditions used here included those that promote the secretion of theseproducts in primary hepatocyte cultures (Be"langer et al. 1978).

Fibronectin was detectable in the cultures of both the undifferentiated cells andtheir retinoic-acid-treated derivatives. Although there are some human teratoma celllines that do not synthesize fibronectin, our observations confirm previous studies onboth uncloned Tera-2 and the NTera-2 clones; it is also known that mouse embryonalcarcinoma cells synthesize and secrete this molecule (Wolfe, Mautner, Hogan & Tilly,1979; Hogan, 1980; Andrews, 1982; Mcllhinney & Patel, 1983; Cossu & Warren,1983; Andrews et al. 1984). The high molecular weight form of fibronectin is secretedby human teratoma lines, primary human teratomas, amniotic epithelial cells andmouse embryonal carcinoma cells; in the case of both the human and mouse teratoma


cells the high molecular weight is attributed to the covalent linkage of fibronectin tolactosaminoglycans and heparan sulphate, but it is also possible that the differentialsplicing of fibronectin messenger RNA might contribute to this shift in apparentmolecular weight (Crouch et al. 1978; Alitalo et al. 1980; Ruoslahti et al. 1981;Mcllhinney & Patel, 1983; Cossu, Andrews & Warren, 1983; Schwarzbauer, Tamku,Lemischka & Hynes, 1983; Kornblihtt, Vibe-Pedersen & Baralle, 1984). Whateverthe explanation is for the decrease in the molecular weight of fibronectin, it is clearthat one of the secreted proteins produced by Tera-2 clones 5, 12 and 13 changes ondifferentiation.

Markers of neurons

A number of reagents have been shown to react with human neurons in culture.These include tetanus toxin, anti-Thy-l(F15.42.1), anti-ganglioside GQlc (F12A2BS) and the monoclonal antibody U13A. However, neurofilament proteins are theonly apparently unequivocal markers of neurons used in this study (see Table 1). Thepresence of cells with long branched processes that react with the two anti-neurofilament antibodies BF10 and RT97 (Anderton et al. 1982) strongly suggeststhat neuron-like cells are formed in response to aggregation and retinoic acid treat-ment. These cells are also neuron-like in their reaction with the reagents F12 A2B5,tetanus toxin and F15.42.1. By analogy with studies on the rat optic nerve, theabsence of staining with anti-GFAP-excludes the possibility that the cells with longprocesses are fibrous astrocytes (Raff et al. 1983). Further, in cell cultures derivedfrom human foetal brain the antibody MIN 1 stains a subpopulation of astrocytes(Dicksone* al. 1982, 1983), and this antibody did not react with the cells bearing longbranching processes in these Tera-2 cultures. Cells with this appearance did not stainwith the 04 antibody which marks oligodendrocytes. Although this antibody has beenfound to label galactocerebroside-positive cells in foetal human brain cultures (G.Dickson, personal communication), previous studies on the mouse nervous systemexclude the possibility that the branched cells represent a type of oligodendrocyte(Somner & Schachner, 1981).

The undifferentiated cells did not express the morphological or antigenic charac-teristics of neurons that we have studied. Upon aggregation and treatment withretinoic acid, these undifferentiated cells give rise to a population in which some cellsexpress a neuron-like morphology and markers, which in the case of neurofilamentsare restricted to neurons. This phenotype was stable over a period of weeks. However,it still remains to be determined whether these cells have the electrical and neurotrans-mitter characteristics of neurons, and also whether it is possible to direct the cellstowards glial differentiation.

The conclusions to be drawn from this study are as follows. (1) The observationthat cloned cells from the Tera-2 human teratoma line can form neuron-like cells hasbeen confirmed (Andrews, 1984), and we have extended the range of antigenic deter-minants that these cells share with human neurons in culture. (2) The capacity to formneuron-like cells can reside in an undifferentiated cell population that is characterizedby a lack of expression of HLA-A,B,C, /82-microglobulin and SSEA-3 determinants.


The majority of the cells express Thy-1, a 90 X \$Mr protein recognized byF10.44.2, and about a third of the cells express the SSEA-1 sugar specificities.(3) Undifferentiated cell populations can be induced to form neuron-like cells undercontrolled experimental conditions. However, there are also clones available that'spontaneously' form these cells. (4) The phenotype of the undifferentiated cells ofthese clones described in this paper closely resembles that of uncloned Tera-2 cul-tures. (5) The undifferentiated cells can develop into populations of static cells inmonolayer that have a distinct, but heterogeneous morphology. They can also developinto cell populations with neuron-like cells. They are therefore a stem cell of thesecultures and they are developmentally at least bipotential.

These studies were generously supported by the Cancer Research Campaign, the MedicalResearch Council and the Muscular Dystrophy of Great Britain Research Fund. We particularlythank Dr Peter Andrews for introducing us to the Tera-2 system and for telling us of his unpublishedresults. We are indebted to Ms L. Johnson and Dr D. W. Mason for assistance and advice with theFACS analysis; the machine was made available by Dr A. F. Williams, MRC Cellular ImmunologyUnit, Oxford. Dr Engstrom was on sabbatical leave from the Department of Tumour Pathology,Karolinska Institutet, Stockholm, supported by travel funds from the Karolinska Institutet andBristol-Myers. Dr Shi was on sabbatical leave from the Shanghai Institute of Cell Biology,Academia Sinica, China, supported by the Royal Society: Chinese Academy of Sciences exchangescheme and the Henry Lester Fund.

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(Received 14 May 1984 -Accepted 8 June 1984)

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