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Mouse teratocarcinoma
Printed in Sweden Copyright @ 1977 by Acadrmic Press. Inc. A// rights of reproduction in any form resewed ISSN 00144827
Experimental Cell Research 105 (1977) 39-50
MOUSE
TERATOCARCINOMA
Carbon Source Utilization Patterns for Growth and In Vitro Differentiation P. AVNER, P. DUBOIS, J. F. NICOLAS, H. JAKOB, J. GAILLARD Service de GPnPtique cellulaire du CollPge de France et de l’lnstitut 75015 Paris. France
and F. JACOB Pasteur,
SUMMARY Carbon source utilization patterns for both in vitro growth and differentiation of the embryonal carcinoma line PCC3/A/l have been investigated. Whilst a number of carbon sources can support growth, the presence of glucose appears critically necessary for in vitro differentiation to reach completion. In the absence of glucose, differentiation is apparently blocked during the second part of the differentiation period. This observation has allowed the isolation of cell variants from PCC3/A/l. Some of the results of a preliminary characterization of such cell lines are discussed.
A number of permanent cell lines of embryonal carcinoma (EC cells) have been established in culture either from the embryoid bodies of transplantable mouse teratocarcinomas or from the solid tumours themselves [l-5]. The majority of these lines do not differentiate in vitro but are capable of differentiating when reinjected in vivo [3,4]. Recently, however, a number of EC cell lines have been reported which can either differentiate in vitro or be maintained in the undifferentiated state depending on the particular culture conditions used [5-g]. Little is presently known about the culture conditions necessary for this in vitro differentiation other than it apparently requires a “critical mass” of cells [6]. This paper describes an investigation into the carbon source utilization patterns of EC cells for both in vitro growth and differentiation. Differentiation was observed to be blocked in the absence of glucose. This ob-
servation suggested a possible method for the generation and isolation of novel cell variants. Some preliminary results of the characterization of the cell lines isolated using this technique are reported.
MATERIALS
AND METHODS
Isolation and characteristics of the embryonal cuvcinoma cell lines used Tumours of the cell lines PCCCF and PCC3/A/l contain differentiated derivatives of all three germ layers, whilst F9 tumours are composed solely of embryonal carcinoma. Only PCC3/A/l is capable of in vitro differentiation [6]. For further details of their isolation and characterization, see Jakob et al. [4].
Stock cultures of the three strains were cultivated on Eagle’s medium (Dulbecco modification) [9] containing 15% foetal calf serum. The cultures were grown in a humid air/CO, mixture at 37°C on Falcon tissue culture dishes. F9 was grown on gelatinized dishes. For further details, see Jakob et al. [4]. All the carbon sources used were dissolved in distilled water and the pH adjusted to 7.4, the solutions being sterilized by Millipore filtration.
Stock cultures.
Exp
Cell
Res 105 (1977)
40
Avner et al.
Growth curves: curbon substrate experiments. All these experiments were performed using as basal medium Dulbecco’s modified Eagle’s medium from which both the glucose and pyruiate, normally present, were omitted (Eagle’s omission medium) and containing 2 % foetal calf serum. To this, the various carbon sources to be tested were added as noted in the text. For any one experiment, media belonging to the same batch and the same bottle of serum were always used. Cells were plated out at densities of 1-3~ lo5 washed cells per 60 mm dish and the growth of the cultures followed using a Coulter counter. The medium was changed every 48 h. Both positive (+glucase) and negative (serum alone) controls were included for each individual series of growth curves. Differentiation cultures: confluence system. In these exoeriments 2x IO6 PCC3IAIl cells were mated uer 60’mm tissue culture dish on Eagle’s omission medium containing 5% foetal calf serum. The medium was changed every 2-3 days during the 28-day differentiation period. The carbon sources added to this medium are noted in the text [6]. Differentiation
cultures:
cell
aggregate
system.
3~ IO6washed PCC3/A/l cells were centrifuged for 3 min at low speed, then incubated at 37°C for 15 min. The pellet was then gently dispersed by pipetting and the resulting variably sized aggregates plated outonto Falcon 1042Petri dishes containing the various media to be tested. Under these conditions a sizeable proportion of the aggregates do not attach to the Petri dish surface but stay in suspension and increase in size. The medium was changed every 48 h. At various times, ranging from 1 to 8 days after their formation, the aggregates were allowed to attach by transferring them to tissue culture dishes (Falcon). The growth media was kept constant in nature and changed every 2-3 days. The differentiation of these aggregates was followed over some 14 days.
Karyology, turnout- formation and histology The methods used have been described previously by Jakob et al. [4].
Immunology Cells from 0 day cultures were used directlv after they had been dissociated with EDTA and washed. Twenty-eight day differentiation cultures were first disaggregated with trypsin-EDTA, then replated out for 24 h before being treated as the above cells [6]. 106cells were incubated for 1 h at 4°C with 50 ~1 of the serum dilution to be tested, dilutions being made in Eagle’s medium containing 4% IPT (Immune Precipitate Tested serum). After washing three times with Eagle’s 4% IPT, the cells were incubated for a further hour at 4°C with F9 absorbed (I/ 1; serum volume : cell volume) fluorescein-labelled RAMIG serum (Rabbit Anti Mouse IgG). The cells were then washed three times with Eagle’s 4% IPT, smears prepared and air-dried, then fixed with methanol and mounted in glycerol/PBS (Phosphate-Buffered Saline). The anti-F9 serum, whose preparation
Immunojluore.scence.
and properties have already been described 1101, was used-at a dilution of l/l00 161.The serum was not absorbed with strain PYS prior to usage. The anti H-2b serum used (F.ASNxB 10.D2 anti B lO.R.5). obtained from Dr R. Motta, was absorbed with F9 (l/4; serum volume : cell volume) prior to usage at a dilution of l/20. Absorptions. H-2 absorptions were carried out with serum diluted l/200 in Hanks’ +4 % IPT using a ratio of l/4, cells to serum. F9 absorptions were performed using a serum dilution of 1 : 600 and a ratio of cells to serum of l/4. All incubations were for 40 min at 4°C and the sera were retested after absorption by direct cytotoxicity tests on lymphocytes and F9 respectively [ 10, 111.
Histochemistry Cells for histochemistry were washed once in PBS, treated in situ for 15-30 min at room temoerature with 1.7% glutaraldehyde in 0.2 M cacodylate buffer pH 7.4, then rewashed three times with PBS. Cells not used immediately were stored at 4°C. Cultures of 0 day cells were fixed directlv 24 h after ulaiting out. Twenty-eight day differentiation cultures, dissociated with trypsin, were replated out at 1x 106 cells/60 mm tissue culture dish, incubated for 12-20 h to allow reattachment, then fixed as above. This procedure avoided the problems of working with multilayered confluent cultures. Alkaline phosphatase (alkaline phos.). Cultures were incubated for 30-60 min at room temperature in 0.2 M Tris buffer pH 8.3 containing 50 pg/ml naphthol AS-MX phosphate and 1 mg/ml Fast Red TR [ 121.The appropriate controls for non-specificity were made and kidney sections treated as the positive control. Acid phosphatase (ucid phos.). Cultures were incubated for 31 h at 37°C in 0.1 M acetate buffer pH 5.3 containing 50 pg/ml naphthol AS-TR phosphate; 1 mgl ml Fast Red TR and several drous of 10% MnCl, solution/40 ml [ 121.The necessary controls for non-specificity and kidney sections as positive controls were made. P-Glucuronidase. The method of Hayashi et al. [ 131 was used. Incubations were carried out at 37°C for 45 min. Kidnev sections were the oositive controls. Ouabain-&sensitive ATPasi. The method of McClurkin 1141was used with the modification that the lead phosphatase ammonium sulphide conversion step was replaced by the use of the rhizonate procedure developed by Meyer [15]. Ouabain when added was used at a final concentration of 0.33 M. Chemicals. All the histochemical substrates used were obtained from Koch-Light, with the exception of the sodium rhizonate, which was obtained from Sigma. The fluorescein-labelled rabbit anti-mouse IgG was obtained from the Institut Pasteur Production Department. The sugars used were obtained from the following suppliers: o+-galactose and sucrose, Prolabo; o+-glucosamine HCI, Calbiochem; o+-mannose, Merck; o--melibiose, Fluka A.G.; o+-fucose, Nutritional Biochemical Corporation, Cleveland; o+-rhamnose, Pfanstiehl Laboratories, Illinois.
Fixation.
In vitro differentiation
41
PCC3/A/l has been maintained on Eagle’s medium containing 2% serum and pyruvate but without glucose for periods of up to 3 months showing that pyruvate at least is capable of supporting growth in long term as well as short term experiments.
‘ime (hours); ordinure: cell countsx 105. Characterization of the serum concentration permitting observation of the stimulatory growth effects of glucose on PCC3/A/l. 1, Eagle’s omission medium alone; 2, Eagle’s omission medium+2% serum; 3, Eagle’s omission medium+2% serum+glucose (2X 10m2M); 4, Eagle’s omission medium+S% serum; 5, Eagle’s omission medium+5% serum+glucose (2x 10mzM). Fig. 1. Abscissa:
In vitro differentiation of PCC3lAll on complete Eagle’s medium As previously described by Nicolas et al. [6], when PCC3/A/l is plated out and left in situ under the conditions outlined in Materials and Methods, a number of events occur leading to the progressive appearance of diverse types of differentiated cells and the concomitant disappearance of EC cells, during the following 28 days. Three or 4 days after plating out, when the culture reaches confluence, triangular flattened epithelial forms appear which are highly contact inhibited. These can either form hollowed out spherical vesicles which I
.
RESULTS Carbon source dependencies for growth of the EC cell lines F9, PCC4-F and PCC3 IAll Preliminary experiments with PCC3/A/l showed that the growth-stimulating effect of glucose could be identified when using 2% fetal calf serum (fig. 1). All growth carbon source dependency experiments were therefore carried out using this concentration. Table 1 and fig. 2 show that both pyruvate and glucose will support the growth of all three EC strains tested. Galactose also supports the growth of PCC3/A/l as do certain, but not all of the tricarboxylic acid intermediates tested (table 1). In so far as they have been tested the responses of the cell lines F9 and PCCCF parallel those of PCC3/A/l (table 1).
L
0
1
,
IO
20
I
30
40
I
50
60
I
70
Fig. 2. Abscissa: time (hours); ordinate: cell countsx 105. The effects of various carbon sources on the growth of PCC3/A/I. O-O, Glucose 2x10-* M; D-m, pyruvate 5~ 10m4M; A-A, isocitrate 2~ IO-* M; O-O, omission medium 2% serum only; X-X, succinate 2~ 10m2M. Exp Cdl Res 105 (1977)
42
Avner et al.
Table 1. Growth response of 3 lines of embryonal carcinoma cells to various carbon substrates Comparison to early stages of embryonic development Capacity to support in vitro growth and development Growth response Carbon source
F9
PCCCF
PCC3/A/l
Mouse 2-celled wx
Glucose Pyruvate Succinate Citrate Isocitrate Malate Acetate Galactose
++++ ++ 0 NT NT NT NT NT
++++ + 0 NT NT NT NT NT
++++ ++ 0 + ++ + 0 ++
0” +a* 0” 0” 0” 0” 0” NT
Mouse 8-celled morula
Rabbit blastocyst
+nb +nb 0” +” NT +ob +a 00
+ + 0 + + + 0 +
The growth response was evaluated using a scale from + + + + to 0 taking the growth on omission media+2 % serum+glucose as the maximal response + + + + , and the growth on omission medium+serum alone as 0 (concentrations used as in table 2). Comparative data on the mouse 2-celled egg and I-celled morula concern the ability of the carbon sources to support the in vitro development of these stages to that of the blastocyst. a Data from Brinster 116. 171. * Data from Whitten [‘18]. The comparative data on the rabbit blastocyst concern the ability of the carbon source to support the in vitro growth of more than 50% of the treated blastocvsts during 24 h. Data from Daniels [19]. Substrate concentrations used were 5X 10e3M. 0, Unable to support development/growth. + , Able to support development/growth. NT, Not tested.
frequently detach from the dish, or transform into extremely large epithelial cells having several nuclei. These cells closely resemble endodermal cells and will be referred to as such [6]. After a period of partial lysis lasting from day 3 to day 7, areas of nervous tissue appear between days 12 and 15. This is in turn followed around days 16-17 by the appearance of unicellular contractile cells (myocarde) and around days 22-24 by the formation of cartilaginous zones. Adipose cells are seen for the first time around day 24 and their appearance is closely followed by that of zones of keratin as well as by the formation of areas of contractile skeletal muscle and pigmented epithelial cells. Thus, in the presence of complete Eagle’s medium, a reproducible Exp Cd Res 105 (IY77)
pattern of in vitro differentiation of PCC3/ A/l occurs within about one month. Effect of carbon source substitution of Eagle’s medium on in vitro differentiation Morphological studies. Table 2 shows the results obtained when the differentiation experiments were carried out on Eagle’s medium in which the glucose and pyruvate had been substituted by other carbon sources. The normal complete in vitro differentiation sequence of PCC3/A/l is supported by only two of the substrates tested, glucose and mannose. These will be referred to as permissive substrates (table 2). All the other carbon sources tested were unable
In vitro differentiation
43
Table 2. Effect of carbon source on the growth and differentiation of PCC3IAIl Carbon substrate
Types of differentiation observed Effect on growth after 28 days (2 % serum) (in the presence of 5 % serum)
Permissivity of carbon source
m-Glucose
++++
Permissive
o+Mannose sf;r;~nnose)
+ or0
Cartilage, keratin, lipid cells, muscle, nerve, etc. Dishes multilayered and non contact inhibited Cartilage, keratin, lipid cells, muscle, nerve, etc. As for glucose Endodermal only NT Endodermal predominantly Cells tend to be either EC or fibroblast-like. No endoderm and no terminal differentiation Endodermal only
Non-permissive Non-permissive
Endodermal only
Non-permissive
Endodermal Endodermal Endodermal Endodermal Endodermal Endodermal Endodermal
Non-permissive Non-permissive Non-nermissive Non-permissive Non-permissive Non-permissive
Glucosamine L-Rhamnose (L-Mannomethylose) o+-Galactose o+-Mehbiose (p a DGalactosido-
++ 0
Permissive Non-permissive Non-permissive
D-Glucose)
D+-Pucose ((YD-Galactomethylose) Succinate Pyruvate Citrate Isocitrate Malate Acetate No added carbon source
- or0 ++
++ + - or0 0
mainly mainly mainlv mainly mainly mainly mainly
The effect on growth was assessed on a scale where + + + + was the response seen in the presence of glucose, 0 was that seen on omission medium+serum without added carbon source. - indicates growth inhibition. NT, not tested. The substrate concentration used was 2~ 10m2M throughout except for pyruvate and citrate when concentrations of 5x IO-* M and 2x 10m3M were used respectively.
to support the full differentiation cycle and will be referred to as non-permissive substrates. Long-term cultures of PCC3/A/l in the presence of non-permissive substrates do not, however, remain as undifferentiated EC cell populations. They are characterised by the formation of predominating cell types differing from those seen with permissive substrates. Non-permissive substrates themselves influence which predominating cell type will be formed. In the presence of galactose, for example, the cell type mainly seen is fibroblast-like. No endoderm is formed and the cells show a marked orientation and alignment. Hata-
naka [20] has noted a similar tendency for galactose to cause cell orientation and has referred to it as contact promotion. Pyruvate on the other hand leads to the formation of endodermal cells as the predominating cell type in 2%day cultures. Experiments were undertaken to test whether the non-permissive substrates were inhibitory to the differentiation sequence supported by permissive substrates such as glucose. Table 3 shows that the differentiation sequence supported by permissive substrates is unaffected by the presence of the non-permissive substrate, all the terminal differentiation markers being formed in such mixtures. E.rp Cd Rr.\ 105 (1977)
44
Avner et al.
Table 3. Effect of pairwise combinations of various carbon sources on the differentiation of PCC3lAll in long term cultures Carbon source
Type of differentiation observed after 28 days
Glucose Pyruvate Sucrose Galactose Glucose+pyruvate Glucose+galactose Glucose+sucrose
Complete differentiation Endoderm Endoderm Fibroblast-like Complete differentiation Complete differentiation Complete differentiation
“Complete differentiation” signifies the presence of nerve, cartilage, pigmented epithelial cells, keratin, endodermal and lipid cells.
Furthermore, the dominant morphological forms characterising the differentiation on a particular non-permissive substrate are suppressed when glucose is present with the non-permissive substrate. The fibroblast-like forms characterizing galactose cultures are not seen in galactose+ glucose cultures. Non-permissive substrates do not therefore seem to inhibit the differentiation process. It is the presence or absence of permissive substrates which is the controlling factor. The effect of carbon source substitution was also studied in the aggregate differentiation system described by Nicolas et al.
[6]. This system allows the formation of nerve to be studied easily, whilst in the confluence system nerve is formed only irregularly. Glucose, pyruvate and sucrose, as well as serum alone all allowed the formation of massive zones of nervous cells. They similarly allowed the formation of the two earliest differentiated forms seen in this system, flattened cells occupying the peripheral zones around the original EC aggregates and endoderm. As in the confluent system only glucose allowed the formation of the later differentiation markers such as cartilage etc. Antigen marker studies. Differentiation of PCC3/A/l on complete medium containing glucose is known to be accompanied by changes in the presence of two antigenic markers: the embryonic antigen F9 on the surface of EC cells decreases, while the histocompatibility locus antigen H-2 increases [6]. When cultures differentiated in the absence of glucose were examined for their antigenic distribution clear differences were found between these cultures and those differentiated in the presence of glucose (table 4). Evidence that the former do not remain as undifferentiated EC cell populations is provided by the marked dimi-
Table 4. Percentage cells from 28 day cultures of PCC3/A/l differentiated in the presence or absence of glucose expressing the F9 and H-2 antigens as detected by immunojluorescence % Cells marked
Antiserum
O-day culture complete medium
28-day culture omission medium +pyruvate (no glucose)
28-day culture omission medium +glucose
Anti-F9 Anti-H2b
50 1
25 17
11 50
O-day culture included as control. For details of sera, see text. Exp (‘e/l /?r.\ 105(1977)
In vitro differentiation
45
Table 5. Histochemical analysis of the enzyme composition of O-day “undifferentiated” and 28-day differentiated cultures of PCC3lA/l Carbon source used in growth/differentiation media
Cell population tested
Enzyme activity tested % positive cells Alkaline phosphatase
Acid phosphatase +=100
ATPaseOuabain insensitive
O-day
Glucose
28-day
Glucose Sucrose
++++= ++++=
5 l- 5
+++=100 NT
Galactose
++++=
5-10
+= 2o 1 100 NT 80 ++++=
+++=100
0 ++=15-30 ++=60
P-Glucuronidase +=100 2- 5 ++++= ++= 90-95 ,w 5-10 I ++++= ++= 80-85 ,@) ++++= 5-15 I
Enzyme activity was detected by histochemical coloration, and assessed on a scale O++ + + + NT, not tested.
ferentiated under different conditions are summarized in table 5. The markers studied were the following four enzymes: alkaline phosphatase, ouabain-insensitive ATPase and the lysosomal enzymes p-glucuronidase and acid phosphatase. All four enzymes show marked changes during in vitro differentiation. In agreement with
nution in the percentage of cells marked by the anti-F9 serum compared to the zero day PCC3/A/I population which consists almost exclusively of EC cells, and the increased percentage of cells marked by the anti-H-2b serum. Histochemical studies. The results of the histochemical characterization of cells dif0 l
+Glucose
,
‘26-30 days Complete dlf
>
60 days NodIf
0 --Glucose 11 days
0 -
I -
+Glucose
-
-Glucose 22 days
0 -
+Glucose
-Glucose
26 days t------
+Glucose
60 days Nodtf
-
,
a + No dlf
0
Fig. 3. Diagrammatic representation of the results obtained with PCC3/A/l from transfer experiments involving shifts from glucose deficient + glucose supplemented media. A 4, see text; F, independence of the time required for the appearance of terminal differentiation markers in glucose supplemented medium from the length of the preculture period in glucosedeficient medium.
-Glucose
Complete
I
30 days Complete
dlf
30 days +
-
-
u
60 days Complete
dlf
dlf.
3 days
28 days
No daf 5 days
26 days
No dlf 25 days
-4
dlf
Complete
1 djf
Complete
1 dlf
29 days No dlf
-Glucose
Complete 23 days
+A-+--
.
dlf
-+
No dlf
I
I Complete
+Glucose
~ E.rp Cd/
Hr.,
10.5 (/977)
46
Avner et al.
Table 6. Preliminary All differentiated
characterization of clonal lines derived from 28 day cultures in the absence of glucose
of PCC3I
Data on cells PCC3/A/I and PCDl included for comparison
Cell line
Carbon source used in isolation
PCC3/A/l/DGI
Galactose
PCC3/A/l/D-G2
Morphology
Tumour type
Galactose
Fibroblastlike Epithelial
Trigerminal teratocarcinoma Trigerminal teratocarcinoma
PCC3/A/l/D-G3
Melibiose
EC like
PCC3/A/l/D-G4
Pyruvate
EC like
PCC3/A/l/D-GS
Pyruvate
Very large epithelial cells
Trigerminal teratocarcinoma Trigerminal teratocarcinoma Non tumourigenic
In vitro differentiation
Chromosome number
Complete differentiation Poorly differentiating mainly cartilage Endoderm only
65-70
Endoderm only
40
No differentiation
70
70-75
39.40
Control lines PCC3/A/l
EC
Trigerminal teratocarcinoma
Complete differentiation
40
PCDl
Fibroblastic (myocardial derivative)
Non tumourigenic
No differentiation
69-77
Complete differentiation indicates that nerve, cartilage, keratin, pigmented epithelial cells, lipid cells and endoderm were all formed. a Tested by absorption. b Tested by immunofluorescence. c Tested by absorption and immunofluorescence. d Tested by absorption, immunofluorescence and cytotoxicity test.
Bemstine et al. [21], it was observed that high alkaline phosphatase activity is present in EC cells, 100% of the cells at day 0 having more or less intense alkaline phosphatase activity. In contrast, the majority of cells from differentiated (permissive) cultures were negative though a subset of 510% were marked even more intensively than the day 0 EC cells. This 5-10% of the cell population are not residual undifferentiated EC cells as the positive cells do not have the antigen F9 and some of them are H-2b positive (P. Avner, in preparation). Exp Ccl/ Res IOS (1977)
The acid phosphatase reaction is positive on 28-day glucose differentiated cells but not on undifferentiated EC cells. Ouabaininsensitive ATPase activity, absent from EC cells, is present in a minor fraction of the 28-day glucose differentiated cell population. p-Glucuronidase activity was weakly present on undifferentiated EC cells and declines overall during differentiation. A minor fraction of some 2-5 % of these cells, however, actually show markedly more activity than that shown by EC cells. The patterns of histochemical reactions
In vitro differentiation
Histochemical test Percent positive cells
Serology Percent positive cells expressing
Acid Alkaline phosphatase phosphatase
H-2 antigen
F9 antigen
0
0
0'
0"
0
N.T.
0’
OC
100
NT
OC
+a
0
0
0*
NT
0
0
NT
0’
100
0
0'
50'
0
100
+”
0’
shown by cultures differentiated on nonpermissive carbon sources show clear differences not only from that of undifferentiated EC day 0 cultures, but equally from the day 28 cultures differentiated in the presence of glucose (table 5). This result supports the conclusion drawn from morphological studies that in the presence of nonpermissive substrates the cells do not remain as undifferentiated EC cells, but equally do not complete the habitual differentiation sequence. The critical period for glucose during differentiation Experiments were designed to detect an eventual block in the differentiation pro46771814
47
cess in the absence of glucose. Two types of experiment were performed: (1) cultures were incubated in the presence of glucose and then switched to medium without glucose; (2) cultures were incubated without glucose for various periods before being switched to a glucose containing medium. Fig. 3 summarizes the results obtained from such transfer experiments. No terminal differentiation occurs when glucose is provided only during the first 11 days prior to transfer into medium lacking glucose (fig. 3~). Glucose must therefore be necessary for differentiation later than the 11th day. This correlates with the fact that early differentiation steps do not seem to be blocked since such forms as endoderm are present on dishes grown without glucose. In contrast, cells which had grown for 22 days in the presence of glucose but which had not yet formed the terminal differentiation markers, when transferred to medium without glucose, continued to differentiate and gave a standard differentiation some 5 days later (fig. 3d). It seems therefore that there is a critical period for the glucose requirement which lies between the 1lth and 22nd day of the differentiation sequence. Controls with already differentiated cultures showed that the differentiated elements once formed are insensitive to glucose deprivation, neither degradation nor necrosis being observed (fig. 3 e). When cells were grown for various periods in the absence of glucose prior to transfer to medium containing glucose, the time required for differentiated forms to become apparent was independent of the time spent in the absence of glucose and corresponded to that found for cultures established continuously in glucose (i.e., 24-28 days) (fig. 3J). It seems therefore that in cultures grown in the absence of glucose, cells do not accumulate directly behind an Erp
C‘rll
f?e.\ /m
(IY77)
48
Avner et al.
11 day block ready to restart differentiation the moment glucose is restored. Isolation of clonal cell lines from 28-day cultures of PCC3/A/l differentiated in the absence of glucose The previous experiments suggested that it might be possible to generate novel cell types, perhaps equivalent to certain intermediary stages in the differentiation process, from cultures differentiated in the absence of glucose. A number of clonal cell lines have thus been isolated from 2%day cultures of PCC3/ A/l differentiated on Eagle’s omission medium, to which had been added, depending on the cell line considered, either galactose, pyruvate or melibiose. Cells were isolated from such cultures after dissociation with trypsin and were cloned on complete Eagle’s medium. The results of a preliminary characterization of these lines is shown in table 6. The majority of the cloned lines tested have retained their tumorigenicity forming trigerminal tumours. These cell lines have therefore remained pluripotential. In this they behaved differently from the majority of cell lines isolated up until now after in vitro differentiation in the presence of glucose (F. Nicolas, unpublished results). The possibility that the formation of trigerminal tumours is due to minor contamination of the strains with EC cells remaining even after cloning would seem to be excluded by the results of a karyotypic analysis of the tumours derived from strain PCC3/A/l/D-Gl. This showed the tumour cells to be aneuploid like this cell line and unlike the EC cells of the parent line PCC3/ A/l. A series of intra-testicular injections made with the cell line PCC3/A/l/D-Gl also showed that trigerminal tumours were formed after injection of as few as ten cells, &I Cell/?e.s 105(IY77)
a result supporting equally the idea that the formation of such tumours is not due to EC cell contamination. The ability of the majority of the cell lines isolated using this selection procedure to form trigerminal tumours and the absence of expression of histocompatibility locus antigens H-2 in vitro from all the lines tested so far suggests that these cell lines are closely related to EC cells. They differ from the latter however in that: (1) the majority of the lines are aneuploid having a chromosome content of 60-70. The strain PCC3/A/l from which they were derived has 40 chromosomes and a relatively normal karyotype [4]. (2) Many of the cell lines have a morphology distinctly different from that of EC cells. Typically, intercell boundaries are more distinct, the cells more contact-inhibited and the nuclear/cytoplasm ratio lower than for EC cells. (3) Many of the lines have lost either completely or partially the capacity to differentiate in vitro. (4) The majority of these cell lines have lost their alkaline phosphatase activity. Alkaline phosphatase activity has up until now been associated with embryonal carcinoma cells [21] and is present in the parent line PCC3/A/l. The cell lines negative for alkaline phosphatase have not been found to have acid phosphatase activity as is the case with both certain previously described differentiated cell lines derived from teratomas, such as the myoblast and myocarde lines PCDl and PCD2 [22] and some cell lines derived in vitro from glucose differentiated cultures. (5) Some of the cell lines tested lacked the F9 antigen when tested both by fluorescence and absorption techniques. These cell lines are at present under
In vitro differentiation further study and a more detailed description will be presented in due course. DISCUSSION The results presented in table 2 establish that of the carbon source substrates tested only two, glucose and mannose, are capable of supporting the habitual complete terminal differentiation sequence of PCC3/A/l in vitro. These two substrates have been called permissive substrates. In the presence of most of the other substrates tested, endoderm alone among the recognizable terminally differentiated cell types is formed. The ability to support a complete differentiation in vitro is not correlated with the ability to support growth (cf galactose and pyruvate) (table 2) and the requirements for differentiation are therefore apparently more stringent than for growth. The growth responses of the three EC strains can equally be compared with those of various mouse embryonic stages, which are known to undergo changes in carbon source utilization patterns during development (table 1). The EC carbon source utilization pattern does not correspond to that of the mouse 2-celled egg but more closely to that of the 8-celled morula. However, even here there is no exact correlation, Such data as is available for mammalian blastocysts (rabbit) suggests, however, that there is a good correlation with mid-late blastocysts (table 1). Corresponding data for mice late morula and blastocysts is, however, needed to confirm this observation. There is no good evidence to suggest that the effect of the absence of permissive substrates on differentiation is to block differentially the formation of derivatives of the three germ layers, as neither mesoderma1 derivatives such as muscle, carti-
49
lage, etc., nor epithelial derivatives such as pigmented retino-epithelial cells or lipid cells are formed in the absence of glucose. It appears more probable that the inhibition pattern seen is solely related to the chronology of the in vitro differentiation process and reflects the temporal order of appearance of the differentiated cell types along its course. This conclusion is in agreement with the results of the transfer experiments which are most easily explained on the basis that there is a critical period between days 11 and 22 of the differentiation sequence during which the presence of a permissive substrate is obligatory. The role which permissive substrates such as glucose play during the in vitro differentiation of PCC3/A/l and the degree to which the observed effect reflects a direct intervention in this process remains to be established. Such studies will, however, naturally be greatly complicated by the complex nature of the culture medium used until now in such in vitro differentiation studies. The results presented here suggested to us that manipulation of the experimental conditions under which EC cells are allowed to differentiate in vitro, by altering the carbon source, might be a useful method allowing the isolation of novel cell variants; cultures differentiated in the presence of non-permissive substrates might represent a material enriched in early differentiated forms and sufficiently enriched so as to be used for their isolation. The preliminary data obtained indeed suggests that the properties of such cell lines corresponds neither to those of EC cells nor to those of the cell lines isolated up until now from 28-day cultures of PCC3/ A/l differentiated in the presence of glucose. Whilst the exact relationship of such E.rp Cell
Rrs
IO5 (1977)
50
Avner et al.
cell types to those occurring during the normal in vitro differentiation sequence in the presence of glucose remains to be established, their novel properties may well make them a valuable material for the investigation of certain problems such as those of the role of surface antigens in differentiation. It is interesting to note, for example, that the F9 antigen does not appear to be obligatorily present on all totipotent cell lines derived from teratocarcinomas and equally that such cells can be markedly aneuploid and retain their capacity to differentiate. This work was supported by grants from the NIH (CA 16355.01) the Andre Meyer Foundation, the Centre National de la Recherche Scientifique (LA no. 88), the Delegation Generale a la Recherche Scientifique et Technique (no. 73.7.1208) and the Foundation Del Duca. One of us (P. A.) acknowledges the support of EMBO and the Philippe Foundation during this work.
REFERENCES 1. Kahan, B N & Ephrussi, B, J natl cancer inst 44 (1970) 1015. 2. Rosenthal, M D, Wishnow, R M & Sato, G H, J natl cancer inst 44 (1970) 1001. 3. Evans, M J, J embryo1 exp morph0128 (1972) 169. 4. Jakob, H, Boon, T, Gaillard, J, Nicolas, J F & Jacob, F, Ann microbial 124B (1973) 269.
Exp Cd Rrs 105 (1977)
5. Lehman, J M, Speers, W C, Swartzendruber, D E & Pierce, G B, J cell physiol84 (1974) 13. 6. Nicolas, J F, Dubois, P, Jakob, H, Gaillard, J & Jacob, F, Ann microbial 126A (1975) 3. 7. Martin, G R & Evans, M J, Cell 2 (1974) 163. 8. - Ibid 6 (1975) 467. 9. Dulbecco, R E & Freeman, G, Virology 8 (1959) 396. 10. Artzt, K, Dubois, P, Bennett, D, Condamine, H, Babinet. C & Jacob, F, Proc natl acad sci US 70 (1973) 2988. 11. Artzt, K & Jacob, F, Transplantation 17 (1974) 633. 12. Burstone, M S, Enzyme histochemistry and its application in the study of neoplasms. Academic Press, New York (1962). 13. Hayashi, M, Nakajima, Y & Fishman, W H, J histochem cytochem 12 (1964) 293. 14. McClurkin, I T, J histochem cytochem 12 (1964) 654. 15. Pearse, A G E, Histochemistry, theoretical and applied, 3rd edn, vol. 2, p. 730. Churchill, London (1968). 16. Brinster, R L & Thomson, J L, Exp cell res 42 (1966) 308. 17. Brinster, R L, In vitro cultivation of mammalian ova, Advances in the Biosciences, vol. 4, p. 199. Schering Symposium on mechanisms involved in conception, Schering, Berlin (ed G Raspe), Peraamon Press (1969). 18. Whzten, W K, Nature 179 (1957) 1081. 19. Daniels, J C Jr, Exp cell res 47 (1967) 619. 20. Hatanaka, M, Biochim biophys acta 355 (1974) 77. 21. Bernstine, E G, Hooper, M L, Grandchamp, S & Ephrussi, B, Proc natl acad sci US 70 (1973) 3899. 22. Boon, T, Buckingham, M E, Dexter, D L, Jakob, H &Jacob, F, Ann microbial 125B (1974) 13. Received August 2, 1976 Accepted October 7. 1976
Experimental Cell Research 105 (1977) 39-50
MOUSE
TERATOCARCINOMA
Carbon Source Utilization Patterns for Growth and In Vitro Differentiation P. AVNER, P. DUBOIS, J. F. NICOLAS, H. JAKOB, J. GAILLARD Service de GPnPtique cellulaire du CollPge de France et de l’lnstitut 75015 Paris. France
and F. JACOB Pasteur,
SUMMARY Carbon source utilization patterns for both in vitro growth and differentiation of the embryonal carcinoma line PCC3/A/l have been investigated. Whilst a number of carbon sources can support growth, the presence of glucose appears critically necessary for in vitro differentiation to reach completion. In the absence of glucose, differentiation is apparently blocked during the second part of the differentiation period. This observation has allowed the isolation of cell variants from PCC3/A/l. Some of the results of a preliminary characterization of such cell lines are discussed.
A number of permanent cell lines of embryonal carcinoma (EC cells) have been established in culture either from the embryoid bodies of transplantable mouse teratocarcinomas or from the solid tumours themselves [l-5]. The majority of these lines do not differentiate in vitro but are capable of differentiating when reinjected in vivo [3,4]. Recently, however, a number of EC cell lines have been reported which can either differentiate in vitro or be maintained in the undifferentiated state depending on the particular culture conditions used [5-g]. Little is presently known about the culture conditions necessary for this in vitro differentiation other than it apparently requires a “critical mass” of cells [6]. This paper describes an investigation into the carbon source utilization patterns of EC cells for both in vitro growth and differentiation. Differentiation was observed to be blocked in the absence of glucose. This ob-
servation suggested a possible method for the generation and isolation of novel cell variants. Some preliminary results of the characterization of the cell lines isolated using this technique are reported.
MATERIALS
AND METHODS
Isolation and characteristics of the embryonal cuvcinoma cell lines used Tumours of the cell lines PCCCF and PCC3/A/l contain differentiated derivatives of all three germ layers, whilst F9 tumours are composed solely of embryonal carcinoma. Only PCC3/A/l is capable of in vitro differentiation [6]. For further details of their isolation and characterization, see Jakob et al. [4].
Stock cultures of the three strains were cultivated on Eagle’s medium (Dulbecco modification) [9] containing 15% foetal calf serum. The cultures were grown in a humid air/CO, mixture at 37°C on Falcon tissue culture dishes. F9 was grown on gelatinized dishes. For further details, see Jakob et al. [4]. All the carbon sources used were dissolved in distilled water and the pH adjusted to 7.4, the solutions being sterilized by Millipore filtration.
Stock cultures.
Exp
Cell
Res 105 (1977)
40
Avner et al.
Growth curves: curbon substrate experiments. All these experiments were performed using as basal medium Dulbecco’s modified Eagle’s medium from which both the glucose and pyruiate, normally present, were omitted (Eagle’s omission medium) and containing 2 % foetal calf serum. To this, the various carbon sources to be tested were added as noted in the text. For any one experiment, media belonging to the same batch and the same bottle of serum were always used. Cells were plated out at densities of 1-3~ lo5 washed cells per 60 mm dish and the growth of the cultures followed using a Coulter counter. The medium was changed every 48 h. Both positive (+glucase) and negative (serum alone) controls were included for each individual series of growth curves. Differentiation cultures: confluence system. In these exoeriments 2x IO6 PCC3IAIl cells were mated uer 60’mm tissue culture dish on Eagle’s omission medium containing 5% foetal calf serum. The medium was changed every 2-3 days during the 28-day differentiation period. The carbon sources added to this medium are noted in the text [6]. Differentiation
cultures:
cell
aggregate
system.
3~ IO6washed PCC3/A/l cells were centrifuged for 3 min at low speed, then incubated at 37°C for 15 min. The pellet was then gently dispersed by pipetting and the resulting variably sized aggregates plated outonto Falcon 1042Petri dishes containing the various media to be tested. Under these conditions a sizeable proportion of the aggregates do not attach to the Petri dish surface but stay in suspension and increase in size. The medium was changed every 48 h. At various times, ranging from 1 to 8 days after their formation, the aggregates were allowed to attach by transferring them to tissue culture dishes (Falcon). The growth media was kept constant in nature and changed every 2-3 days. The differentiation of these aggregates was followed over some 14 days.
Karyology, turnout- formation and histology The methods used have been described previously by Jakob et al. [4].
Immunology Cells from 0 day cultures were used directlv after they had been dissociated with EDTA and washed. Twenty-eight day differentiation cultures were first disaggregated with trypsin-EDTA, then replated out for 24 h before being treated as the above cells [6]. 106cells were incubated for 1 h at 4°C with 50 ~1 of the serum dilution to be tested, dilutions being made in Eagle’s medium containing 4% IPT (Immune Precipitate Tested serum). After washing three times with Eagle’s 4% IPT, the cells were incubated for a further hour at 4°C with F9 absorbed (I/ 1; serum volume : cell volume) fluorescein-labelled RAMIG serum (Rabbit Anti Mouse IgG). The cells were then washed three times with Eagle’s 4% IPT, smears prepared and air-dried, then fixed with methanol and mounted in glycerol/PBS (Phosphate-Buffered Saline). The anti-F9 serum, whose preparation
Immunojluore.scence.
and properties have already been described 1101, was used-at a dilution of l/l00 161.The serum was not absorbed with strain PYS prior to usage. The anti H-2b serum used (F.ASNxB 10.D2 anti B lO.R.5). obtained from Dr R. Motta, was absorbed with F9 (l/4; serum volume : cell volume) prior to usage at a dilution of l/20. Absorptions. H-2 absorptions were carried out with serum diluted l/200 in Hanks’ +4 % IPT using a ratio of l/4, cells to serum. F9 absorptions were performed using a serum dilution of 1 : 600 and a ratio of cells to serum of l/4. All incubations were for 40 min at 4°C and the sera were retested after absorption by direct cytotoxicity tests on lymphocytes and F9 respectively [ 10, 111.
Histochemistry Cells for histochemistry were washed once in PBS, treated in situ for 15-30 min at room temoerature with 1.7% glutaraldehyde in 0.2 M cacodylate buffer pH 7.4, then rewashed three times with PBS. Cells not used immediately were stored at 4°C. Cultures of 0 day cells were fixed directlv 24 h after ulaiting out. Twenty-eight day differentiation cultures, dissociated with trypsin, were replated out at 1x 106 cells/60 mm tissue culture dish, incubated for 12-20 h to allow reattachment, then fixed as above. This procedure avoided the problems of working with multilayered confluent cultures. Alkaline phosphatase (alkaline phos.). Cultures were incubated for 30-60 min at room temperature in 0.2 M Tris buffer pH 8.3 containing 50 pg/ml naphthol AS-MX phosphate and 1 mg/ml Fast Red TR [ 121.The appropriate controls for non-specificity were made and kidney sections treated as the positive control. Acid phosphatase (ucid phos.). Cultures were incubated for 31 h at 37°C in 0.1 M acetate buffer pH 5.3 containing 50 pg/ml naphthol AS-TR phosphate; 1 mgl ml Fast Red TR and several drous of 10% MnCl, solution/40 ml [ 121.The necessary controls for non-specificity and kidney sections as positive controls were made. P-Glucuronidase. The method of Hayashi et al. [ 131 was used. Incubations were carried out at 37°C for 45 min. Kidnev sections were the oositive controls. Ouabain-&sensitive ATPasi. The method of McClurkin 1141was used with the modification that the lead phosphatase ammonium sulphide conversion step was replaced by the use of the rhizonate procedure developed by Meyer [15]. Ouabain when added was used at a final concentration of 0.33 M. Chemicals. All the histochemical substrates used were obtained from Koch-Light, with the exception of the sodium rhizonate, which was obtained from Sigma. The fluorescein-labelled rabbit anti-mouse IgG was obtained from the Institut Pasteur Production Department. The sugars used were obtained from the following suppliers: o+-galactose and sucrose, Prolabo; o+-glucosamine HCI, Calbiochem; o+-mannose, Merck; o--melibiose, Fluka A.G.; o+-fucose, Nutritional Biochemical Corporation, Cleveland; o+-rhamnose, Pfanstiehl Laboratories, Illinois.
Fixation.
In vitro differentiation
41
PCC3/A/l has been maintained on Eagle’s medium containing 2% serum and pyruvate but without glucose for periods of up to 3 months showing that pyruvate at least is capable of supporting growth in long term as well as short term experiments.
‘ime (hours); ordinure: cell countsx 105. Characterization of the serum concentration permitting observation of the stimulatory growth effects of glucose on PCC3/A/l. 1, Eagle’s omission medium alone; 2, Eagle’s omission medium+2% serum; 3, Eagle’s omission medium+2% serum+glucose (2X 10m2M); 4, Eagle’s omission medium+S% serum; 5, Eagle’s omission medium+5% serum+glucose (2x 10mzM). Fig. 1. Abscissa:
In vitro differentiation of PCC3lAll on complete Eagle’s medium As previously described by Nicolas et al. [6], when PCC3/A/l is plated out and left in situ under the conditions outlined in Materials and Methods, a number of events occur leading to the progressive appearance of diverse types of differentiated cells and the concomitant disappearance of EC cells, during the following 28 days. Three or 4 days after plating out, when the culture reaches confluence, triangular flattened epithelial forms appear which are highly contact inhibited. These can either form hollowed out spherical vesicles which I
.
RESULTS Carbon source dependencies for growth of the EC cell lines F9, PCC4-F and PCC3 IAll Preliminary experiments with PCC3/A/l showed that the growth-stimulating effect of glucose could be identified when using 2% fetal calf serum (fig. 1). All growth carbon source dependency experiments were therefore carried out using this concentration. Table 1 and fig. 2 show that both pyruvate and glucose will support the growth of all three EC strains tested. Galactose also supports the growth of PCC3/A/l as do certain, but not all of the tricarboxylic acid intermediates tested (table 1). In so far as they have been tested the responses of the cell lines F9 and PCCCF parallel those of PCC3/A/l (table 1).
L
0
1
,
IO
20
I
30
40
I
50
60
I
70
Fig. 2. Abscissa: time (hours); ordinate: cell countsx 105. The effects of various carbon sources on the growth of PCC3/A/I. O-O, Glucose 2x10-* M; D-m, pyruvate 5~ 10m4M; A-A, isocitrate 2~ IO-* M; O-O, omission medium 2% serum only; X-X, succinate 2~ 10m2M. Exp Cdl Res 105 (1977)
42
Avner et al.
Table 1. Growth response of 3 lines of embryonal carcinoma cells to various carbon substrates Comparison to early stages of embryonic development Capacity to support in vitro growth and development Growth response Carbon source
F9
PCCCF
PCC3/A/l
Mouse 2-celled wx
Glucose Pyruvate Succinate Citrate Isocitrate Malate Acetate Galactose
++++ ++ 0 NT NT NT NT NT
++++ + 0 NT NT NT NT NT
++++ ++ 0 + ++ + 0 ++
0” +a* 0” 0” 0” 0” 0” NT
Mouse 8-celled morula
Rabbit blastocyst
+nb +nb 0” +” NT +ob +a 00
+ + 0 + + + 0 +
The growth response was evaluated using a scale from + + + + to 0 taking the growth on omission media+2 % serum+glucose as the maximal response + + + + , and the growth on omission medium+serum alone as 0 (concentrations used as in table 2). Comparative data on the mouse 2-celled egg and I-celled morula concern the ability of the carbon sources to support the in vitro development of these stages to that of the blastocyst. a Data from Brinster 116. 171. * Data from Whitten [‘18]. The comparative data on the rabbit blastocyst concern the ability of the carbon source to support the in vitro growth of more than 50% of the treated blastocvsts during 24 h. Data from Daniels [19]. Substrate concentrations used were 5X 10e3M. 0, Unable to support development/growth. + , Able to support development/growth. NT, Not tested.
frequently detach from the dish, or transform into extremely large epithelial cells having several nuclei. These cells closely resemble endodermal cells and will be referred to as such [6]. After a period of partial lysis lasting from day 3 to day 7, areas of nervous tissue appear between days 12 and 15. This is in turn followed around days 16-17 by the appearance of unicellular contractile cells (myocarde) and around days 22-24 by the formation of cartilaginous zones. Adipose cells are seen for the first time around day 24 and their appearance is closely followed by that of zones of keratin as well as by the formation of areas of contractile skeletal muscle and pigmented epithelial cells. Thus, in the presence of complete Eagle’s medium, a reproducible Exp Cd Res 105 (IY77)
pattern of in vitro differentiation of PCC3/ A/l occurs within about one month. Effect of carbon source substitution of Eagle’s medium on in vitro differentiation Morphological studies. Table 2 shows the results obtained when the differentiation experiments were carried out on Eagle’s medium in which the glucose and pyruvate had been substituted by other carbon sources. The normal complete in vitro differentiation sequence of PCC3/A/l is supported by only two of the substrates tested, glucose and mannose. These will be referred to as permissive substrates (table 2). All the other carbon sources tested were unable
In vitro differentiation
43
Table 2. Effect of carbon source on the growth and differentiation of PCC3IAIl Carbon substrate
Types of differentiation observed Effect on growth after 28 days (2 % serum) (in the presence of 5 % serum)
Permissivity of carbon source
m-Glucose
++++
Permissive
o+Mannose sf;r;~nnose)
+ or0
Cartilage, keratin, lipid cells, muscle, nerve, etc. Dishes multilayered and non contact inhibited Cartilage, keratin, lipid cells, muscle, nerve, etc. As for glucose Endodermal only NT Endodermal predominantly Cells tend to be either EC or fibroblast-like. No endoderm and no terminal differentiation Endodermal only
Non-permissive Non-permissive
Endodermal only
Non-permissive
Endodermal Endodermal Endodermal Endodermal Endodermal Endodermal Endodermal
Non-permissive Non-permissive Non-nermissive Non-permissive Non-permissive Non-permissive
Glucosamine L-Rhamnose (L-Mannomethylose) o+-Galactose o+-Mehbiose (p a DGalactosido-
++ 0
Permissive Non-permissive Non-permissive
D-Glucose)
D+-Pucose ((YD-Galactomethylose) Succinate Pyruvate Citrate Isocitrate Malate Acetate No added carbon source
- or0 ++
++ + - or0 0
mainly mainly mainlv mainly mainly mainly mainly
The effect on growth was assessed on a scale where + + + + was the response seen in the presence of glucose, 0 was that seen on omission medium+serum without added carbon source. - indicates growth inhibition. NT, not tested. The substrate concentration used was 2~ 10m2M throughout except for pyruvate and citrate when concentrations of 5x IO-* M and 2x 10m3M were used respectively.
to support the full differentiation cycle and will be referred to as non-permissive substrates. Long-term cultures of PCC3/A/l in the presence of non-permissive substrates do not, however, remain as undifferentiated EC cell populations. They are characterised by the formation of predominating cell types differing from those seen with permissive substrates. Non-permissive substrates themselves influence which predominating cell type will be formed. In the presence of galactose, for example, the cell type mainly seen is fibroblast-like. No endoderm is formed and the cells show a marked orientation and alignment. Hata-
naka [20] has noted a similar tendency for galactose to cause cell orientation and has referred to it as contact promotion. Pyruvate on the other hand leads to the formation of endodermal cells as the predominating cell type in 2%day cultures. Experiments were undertaken to test whether the non-permissive substrates were inhibitory to the differentiation sequence supported by permissive substrates such as glucose. Table 3 shows that the differentiation sequence supported by permissive substrates is unaffected by the presence of the non-permissive substrate, all the terminal differentiation markers being formed in such mixtures. E.rp Cd Rr.\ 105 (1977)
44
Avner et al.
Table 3. Effect of pairwise combinations of various carbon sources on the differentiation of PCC3lAll in long term cultures Carbon source
Type of differentiation observed after 28 days
Glucose Pyruvate Sucrose Galactose Glucose+pyruvate Glucose+galactose Glucose+sucrose
Complete differentiation Endoderm Endoderm Fibroblast-like Complete differentiation Complete differentiation Complete differentiation
“Complete differentiation” signifies the presence of nerve, cartilage, pigmented epithelial cells, keratin, endodermal and lipid cells.
Furthermore, the dominant morphological forms characterising the differentiation on a particular non-permissive substrate are suppressed when glucose is present with the non-permissive substrate. The fibroblast-like forms characterizing galactose cultures are not seen in galactose+ glucose cultures. Non-permissive substrates do not therefore seem to inhibit the differentiation process. It is the presence or absence of permissive substrates which is the controlling factor. The effect of carbon source substitution was also studied in the aggregate differentiation system described by Nicolas et al.
[6]. This system allows the formation of nerve to be studied easily, whilst in the confluence system nerve is formed only irregularly. Glucose, pyruvate and sucrose, as well as serum alone all allowed the formation of massive zones of nervous cells. They similarly allowed the formation of the two earliest differentiated forms seen in this system, flattened cells occupying the peripheral zones around the original EC aggregates and endoderm. As in the confluent system only glucose allowed the formation of the later differentiation markers such as cartilage etc. Antigen marker studies. Differentiation of PCC3/A/l on complete medium containing glucose is known to be accompanied by changes in the presence of two antigenic markers: the embryonic antigen F9 on the surface of EC cells decreases, while the histocompatibility locus antigen H-2 increases [6]. When cultures differentiated in the absence of glucose were examined for their antigenic distribution clear differences were found between these cultures and those differentiated in the presence of glucose (table 4). Evidence that the former do not remain as undifferentiated EC cell populations is provided by the marked dimi-
Table 4. Percentage cells from 28 day cultures of PCC3/A/l differentiated in the presence or absence of glucose expressing the F9 and H-2 antigens as detected by immunojluorescence % Cells marked
Antiserum
O-day culture complete medium
28-day culture omission medium +pyruvate (no glucose)
28-day culture omission medium +glucose
Anti-F9 Anti-H2b
50 1
25 17
11 50
O-day culture included as control. For details of sera, see text. Exp (‘e/l /?r.\ 105(1977)
In vitro differentiation
45
Table 5. Histochemical analysis of the enzyme composition of O-day “undifferentiated” and 28-day differentiated cultures of PCC3lA/l Carbon source used in growth/differentiation media
Cell population tested
Enzyme activity tested % positive cells Alkaline phosphatase
Acid phosphatase +=100
ATPaseOuabain insensitive
O-day
Glucose
28-day
Glucose Sucrose
++++= ++++=
5 l- 5
+++=100 NT
Galactose
++++=
5-10
+= 2o 1 100 NT 80 ++++=
+++=100
0 ++=15-30 ++=60
P-Glucuronidase +=100 2- 5 ++++= ++= 90-95 ,w 5-10 I ++++= ++= 80-85 ,@) ++++= 5-15 I
Enzyme activity was detected by histochemical coloration, and assessed on a scale O++ + + + NT, not tested.
ferentiated under different conditions are summarized in table 5. The markers studied were the following four enzymes: alkaline phosphatase, ouabain-insensitive ATPase and the lysosomal enzymes p-glucuronidase and acid phosphatase. All four enzymes show marked changes during in vitro differentiation. In agreement with
nution in the percentage of cells marked by the anti-F9 serum compared to the zero day PCC3/A/I population which consists almost exclusively of EC cells, and the increased percentage of cells marked by the anti-H-2b serum. Histochemical studies. The results of the histochemical characterization of cells dif0 l
+Glucose
,
‘26-30 days Complete dlf
>
60 days NodIf
0 --Glucose 11 days
0 -
I -
+Glucose
-
-Glucose 22 days
0 -
+Glucose
-Glucose
26 days t------
+Glucose
60 days Nodtf
-
,
a + No dlf
0
Fig. 3. Diagrammatic representation of the results obtained with PCC3/A/l from transfer experiments involving shifts from glucose deficient + glucose supplemented media. A 4, see text; F, independence of the time required for the appearance of terminal differentiation markers in glucose supplemented medium from the length of the preculture period in glucosedeficient medium.
-Glucose
Complete
I
30 days Complete
dlf
30 days +
-
-
u
60 days Complete
dlf
dlf.
3 days
28 days
No daf 5 days
26 days
No dlf 25 days
-4
dlf
Complete
1 djf
Complete
1 dlf
29 days No dlf
-Glucose
Complete 23 days
+A-+--
.
dlf
-+
No dlf
I
I Complete
+Glucose
~ E.rp Cd/
Hr.,
10.5 (/977)
46
Avner et al.
Table 6. Preliminary All differentiated
characterization of clonal lines derived from 28 day cultures in the absence of glucose
of PCC3I
Data on cells PCC3/A/I and PCDl included for comparison
Cell line
Carbon source used in isolation
PCC3/A/l/DGI
Galactose
PCC3/A/l/D-G2
Morphology
Tumour type
Galactose
Fibroblastlike Epithelial
Trigerminal teratocarcinoma Trigerminal teratocarcinoma
PCC3/A/l/D-G3
Melibiose
EC like
PCC3/A/l/D-G4
Pyruvate
EC like
PCC3/A/l/D-GS
Pyruvate
Very large epithelial cells
Trigerminal teratocarcinoma Trigerminal teratocarcinoma Non tumourigenic
In vitro differentiation
Chromosome number
Complete differentiation Poorly differentiating mainly cartilage Endoderm only
65-70
Endoderm only
40
No differentiation
70
70-75
39.40
Control lines PCC3/A/l
EC
Trigerminal teratocarcinoma
Complete differentiation
40
PCDl
Fibroblastic (myocardial derivative)
Non tumourigenic
No differentiation
69-77
Complete differentiation indicates that nerve, cartilage, keratin, pigmented epithelial cells, lipid cells and endoderm were all formed. a Tested by absorption. b Tested by immunofluorescence. c Tested by absorption and immunofluorescence. d Tested by absorption, immunofluorescence and cytotoxicity test.
Bemstine et al. [21], it was observed that high alkaline phosphatase activity is present in EC cells, 100% of the cells at day 0 having more or less intense alkaline phosphatase activity. In contrast, the majority of cells from differentiated (permissive) cultures were negative though a subset of 510% were marked even more intensively than the day 0 EC cells. This 5-10% of the cell population are not residual undifferentiated EC cells as the positive cells do not have the antigen F9 and some of them are H-2b positive (P. Avner, in preparation). Exp Ccl/ Res IOS (1977)
The acid phosphatase reaction is positive on 28-day glucose differentiated cells but not on undifferentiated EC cells. Ouabaininsensitive ATPase activity, absent from EC cells, is present in a minor fraction of the 28-day glucose differentiated cell population. p-Glucuronidase activity was weakly present on undifferentiated EC cells and declines overall during differentiation. A minor fraction of some 2-5 % of these cells, however, actually show markedly more activity than that shown by EC cells. The patterns of histochemical reactions
In vitro differentiation
Histochemical test Percent positive cells
Serology Percent positive cells expressing
Acid Alkaline phosphatase phosphatase
H-2 antigen
F9 antigen
0
0
0'
0"
0
N.T.
0’
OC
100
NT
OC
+a
0
0
0*
NT
0
0
NT
0’
100
0
0'
50'
0
100
+”
0’
shown by cultures differentiated on nonpermissive carbon sources show clear differences not only from that of undifferentiated EC day 0 cultures, but equally from the day 28 cultures differentiated in the presence of glucose (table 5). This result supports the conclusion drawn from morphological studies that in the presence of nonpermissive substrates the cells do not remain as undifferentiated EC cells, but equally do not complete the habitual differentiation sequence. The critical period for glucose during differentiation Experiments were designed to detect an eventual block in the differentiation pro46771814
47
cess in the absence of glucose. Two types of experiment were performed: (1) cultures were incubated in the presence of glucose and then switched to medium without glucose; (2) cultures were incubated without glucose for various periods before being switched to a glucose containing medium. Fig. 3 summarizes the results obtained from such transfer experiments. No terminal differentiation occurs when glucose is provided only during the first 11 days prior to transfer into medium lacking glucose (fig. 3~). Glucose must therefore be necessary for differentiation later than the 11th day. This correlates with the fact that early differentiation steps do not seem to be blocked since such forms as endoderm are present on dishes grown without glucose. In contrast, cells which had grown for 22 days in the presence of glucose but which had not yet formed the terminal differentiation markers, when transferred to medium without glucose, continued to differentiate and gave a standard differentiation some 5 days later (fig. 3d). It seems therefore that there is a critical period for the glucose requirement which lies between the 1lth and 22nd day of the differentiation sequence. Controls with already differentiated cultures showed that the differentiated elements once formed are insensitive to glucose deprivation, neither degradation nor necrosis being observed (fig. 3 e). When cells were grown for various periods in the absence of glucose prior to transfer to medium containing glucose, the time required for differentiated forms to become apparent was independent of the time spent in the absence of glucose and corresponded to that found for cultures established continuously in glucose (i.e., 24-28 days) (fig. 3J). It seems therefore that in cultures grown in the absence of glucose, cells do not accumulate directly behind an Erp
C‘rll
f?e.\ /m
(IY77)
48
Avner et al.
11 day block ready to restart differentiation the moment glucose is restored. Isolation of clonal cell lines from 28-day cultures of PCC3/A/l differentiated in the absence of glucose The previous experiments suggested that it might be possible to generate novel cell types, perhaps equivalent to certain intermediary stages in the differentiation process, from cultures differentiated in the absence of glucose. A number of clonal cell lines have thus been isolated from 2%day cultures of PCC3/ A/l differentiated on Eagle’s omission medium, to which had been added, depending on the cell line considered, either galactose, pyruvate or melibiose. Cells were isolated from such cultures after dissociation with trypsin and were cloned on complete Eagle’s medium. The results of a preliminary characterization of these lines is shown in table 6. The majority of the cloned lines tested have retained their tumorigenicity forming trigerminal tumours. These cell lines have therefore remained pluripotential. In this they behaved differently from the majority of cell lines isolated up until now after in vitro differentiation in the presence of glucose (F. Nicolas, unpublished results). The possibility that the formation of trigerminal tumours is due to minor contamination of the strains with EC cells remaining even after cloning would seem to be excluded by the results of a karyotypic analysis of the tumours derived from strain PCC3/A/l/D-Gl. This showed the tumour cells to be aneuploid like this cell line and unlike the EC cells of the parent line PCC3/ A/l. A series of intra-testicular injections made with the cell line PCC3/A/l/D-Gl also showed that trigerminal tumours were formed after injection of as few as ten cells, &I Cell/?e.s 105(IY77)
a result supporting equally the idea that the formation of such tumours is not due to EC cell contamination. The ability of the majority of the cell lines isolated using this selection procedure to form trigerminal tumours and the absence of expression of histocompatibility locus antigens H-2 in vitro from all the lines tested so far suggests that these cell lines are closely related to EC cells. They differ from the latter however in that: (1) the majority of the lines are aneuploid having a chromosome content of 60-70. The strain PCC3/A/l from which they were derived has 40 chromosomes and a relatively normal karyotype [4]. (2) Many of the cell lines have a morphology distinctly different from that of EC cells. Typically, intercell boundaries are more distinct, the cells more contact-inhibited and the nuclear/cytoplasm ratio lower than for EC cells. (3) Many of the lines have lost either completely or partially the capacity to differentiate in vitro. (4) The majority of these cell lines have lost their alkaline phosphatase activity. Alkaline phosphatase activity has up until now been associated with embryonal carcinoma cells [21] and is present in the parent line PCC3/A/l. The cell lines negative for alkaline phosphatase have not been found to have acid phosphatase activity as is the case with both certain previously described differentiated cell lines derived from teratomas, such as the myoblast and myocarde lines PCDl and PCD2 [22] and some cell lines derived in vitro from glucose differentiated cultures. (5) Some of the cell lines tested lacked the F9 antigen when tested both by fluorescence and absorption techniques. These cell lines are at present under
In vitro differentiation further study and a more detailed description will be presented in due course. DISCUSSION The results presented in table 2 establish that of the carbon source substrates tested only two, glucose and mannose, are capable of supporting the habitual complete terminal differentiation sequence of PCC3/A/l in vitro. These two substrates have been called permissive substrates. In the presence of most of the other substrates tested, endoderm alone among the recognizable terminally differentiated cell types is formed. The ability to support a complete differentiation in vitro is not correlated with the ability to support growth (cf galactose and pyruvate) (table 2) and the requirements for differentiation are therefore apparently more stringent than for growth. The growth responses of the three EC strains can equally be compared with those of various mouse embryonic stages, which are known to undergo changes in carbon source utilization patterns during development (table 1). The EC carbon source utilization pattern does not correspond to that of the mouse 2-celled egg but more closely to that of the 8-celled morula. However, even here there is no exact correlation, Such data as is available for mammalian blastocysts (rabbit) suggests, however, that there is a good correlation with mid-late blastocysts (table 1). Corresponding data for mice late morula and blastocysts is, however, needed to confirm this observation. There is no good evidence to suggest that the effect of the absence of permissive substrates on differentiation is to block differentially the formation of derivatives of the three germ layers, as neither mesoderma1 derivatives such as muscle, carti-
49
lage, etc., nor epithelial derivatives such as pigmented retino-epithelial cells or lipid cells are formed in the absence of glucose. It appears more probable that the inhibition pattern seen is solely related to the chronology of the in vitro differentiation process and reflects the temporal order of appearance of the differentiated cell types along its course. This conclusion is in agreement with the results of the transfer experiments which are most easily explained on the basis that there is a critical period between days 11 and 22 of the differentiation sequence during which the presence of a permissive substrate is obligatory. The role which permissive substrates such as glucose play during the in vitro differentiation of PCC3/A/l and the degree to which the observed effect reflects a direct intervention in this process remains to be established. Such studies will, however, naturally be greatly complicated by the complex nature of the culture medium used until now in such in vitro differentiation studies. The results presented here suggested to us that manipulation of the experimental conditions under which EC cells are allowed to differentiate in vitro, by altering the carbon source, might be a useful method allowing the isolation of novel cell variants; cultures differentiated in the presence of non-permissive substrates might represent a material enriched in early differentiated forms and sufficiently enriched so as to be used for their isolation. The preliminary data obtained indeed suggests that the properties of such cell lines corresponds neither to those of EC cells nor to those of the cell lines isolated up until now from 28-day cultures of PCC3/ A/l differentiated in the presence of glucose. Whilst the exact relationship of such E.rp Cell
Rrs
IO5 (1977)
50
Avner et al.
cell types to those occurring during the normal in vitro differentiation sequence in the presence of glucose remains to be established, their novel properties may well make them a valuable material for the investigation of certain problems such as those of the role of surface antigens in differentiation. It is interesting to note, for example, that the F9 antigen does not appear to be obligatorily present on all totipotent cell lines derived from teratocarcinomas and equally that such cells can be markedly aneuploid and retain their capacity to differentiate. This work was supported by grants from the NIH (CA 16355.01) the Andre Meyer Foundation, the Centre National de la Recherche Scientifique (LA no. 88), the Delegation Generale a la Recherche Scientifique et Technique (no. 73.7.1208) and the Foundation Del Duca. One of us (P. A.) acknowledges the support of EMBO and the Philippe Foundation during this work.
REFERENCES 1. Kahan, B N & Ephrussi, B, J natl cancer inst 44 (1970) 1015. 2. Rosenthal, M D, Wishnow, R M & Sato, G H, J natl cancer inst 44 (1970) 1001. 3. Evans, M J, J embryo1 exp morph0128 (1972) 169. 4. Jakob, H, Boon, T, Gaillard, J, Nicolas, J F & Jacob, F, Ann microbial 124B (1973) 269.
Exp Cd Rrs 105 (1977)
5. Lehman, J M, Speers, W C, Swartzendruber, D E & Pierce, G B, J cell physiol84 (1974) 13. 6. Nicolas, J F, Dubois, P, Jakob, H, Gaillard, J & Jacob, F, Ann microbial 126A (1975) 3. 7. Martin, G R & Evans, M J, Cell 2 (1974) 163. 8. - Ibid 6 (1975) 467. 9. Dulbecco, R E & Freeman, G, Virology 8 (1959) 396. 10. Artzt, K, Dubois, P, Bennett, D, Condamine, H, Babinet. C & Jacob, F, Proc natl acad sci US 70 (1973) 2988. 11. Artzt, K & Jacob, F, Transplantation 17 (1974) 633. 12. Burstone, M S, Enzyme histochemistry and its application in the study of neoplasms. Academic Press, New York (1962). 13. Hayashi, M, Nakajima, Y & Fishman, W H, J histochem cytochem 12 (1964) 293. 14. McClurkin, I T, J histochem cytochem 12 (1964) 654. 15. Pearse, A G E, Histochemistry, theoretical and applied, 3rd edn, vol. 2, p. 730. Churchill, London (1968). 16. Brinster, R L & Thomson, J L, Exp cell res 42 (1966) 308. 17. Brinster, R L, In vitro cultivation of mammalian ova, Advances in the Biosciences, vol. 4, p. 199. Schering Symposium on mechanisms involved in conception, Schering, Berlin (ed G Raspe), Peraamon Press (1969). 18. Whzten, W K, Nature 179 (1957) 1081. 19. Daniels, J C Jr, Exp cell res 47 (1967) 619. 20. Hatanaka, M, Biochim biophys acta 355 (1974) 77. 21. Bernstine, E G, Hooper, M L, Grandchamp, S & Ephrussi, B, Proc natl acad sci US 70 (1973) 3899. 22. Boon, T, Buckingham, M E, Dexter, D L, Jakob, H &Jacob, F, Ann microbial 125B (1974) 13. Received August 2, 1976 Accepted October 7. 1976