Download Pluripotent differentiation of single f9 embryonal carcinoma cells

Short notes
567
SHORT NOTE
Pluripotent
PETER
‘Murdoch
Institute,
Differentiation
KOOPMAN’,
Royal
Children’s
bourne,
of Single F9 Embryonal
*, * and RICHARD
Hospital
Parkville
Carcinoma
Cells
G. H. COTTON’
and ‘Department
of Paediatrics,
3052, Victoria,
Australia
University
of Mel-
F9 embryonal carcinoma cells were induced to form a variety of differentiated cell types
in monolayer culture. Cells with the morphological, histochemical and immunocytochemical properties of parietal and visceral endoderm, neurones and adipocytes were identified.
Cells expressing Thy-l antigen and large, multinucleated cells expressing cytoplasmic
fibronectin were also observed. Various cell types were found together in colonies derived
from individual F9 cells, allowing us to conclude that F9 cells are pluripotent in vitro.
@ 1987 Academic
Press, Inc.
The differentiation
of murine embryonal carcinoma (EC) cells in vitro is
commonly used as a model of developmental events in the embryo [ 1, 21. The F9
EC cell line [3], one of the most extensively studied, shows minimal spontaneous
differentiation [4], but can be induced to differentiate in vitro with agents such as
retinoic acid (RA) [5, 61. However, a restricted range of differentiated
cell
lineages is generated in this way [5-91, which limits the validity and usefulness of
these cells as a model for normal events. We report here that single F9 cells can
be induced to form a variety of differentiated cell types together in low-density
colonies in monolayer culture.
Materials and Methods
Cell culture.
F9 cells [3] from Dr A. Levine, Princeton University, N. J., were grown as previously
described [lo] in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum
(FBS) (Flow Laboratories), in gelatin-coated culture vessels. Retinoic acid (BA) (Sigma, all-trans)
was stored at -20°C as a lo-’ M stock in absolute ethanol, and diluted freshly into culture medium
before use. ST0 fibroblast-conditioned
medium was produced as previously described [IO]. Cell
stocks were found free of mycoplasma by Hoechst 33258 staining [l 11.
ZdentiJication
of cell types. Various antigens were visualized by a modified peroxidase-antiperoxidase
(PAP) technique [12]. Colonies were fixed in 5 % acetic acid/95% ethanol at -20°C for 10 min,
thoroughly washed and incubated in 10 % normal swine serum (Flow) in phosphate-buffered saline for
30 min. Incubation for 1 h with primary antibody was followed by 30 min incubations with rabbit antimouse Ig (Dako 2109) at 1 : 200 where mouse or rat primary antibodies were used, or biotinylated
rabbit anti-sheep Ig (Vector BA 6000) where sheep primary antibody was used, then swine anti-rabbit
Ig (Dako 2196) at 1: 25, and finally rabbit PAP complex (Dako Zl13) at 1: 200. The chromogen was
diaminobenzidine (Sigma). All incubations were at room temperature. In the case of A2B5 and its
negative control antibody, fixation followed primary antibody incubation and washing. Primary
antibodies were as follows: LE61 [13], a mouse monoclonal IgG, antibody supematant recognizing
* To whom offprint requests should be sent. Present address: MRC Mammalian Development Unit,
Wolfson House, 4 Stephenson Way, London NW1 2HE, England.
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568 Short notes
cytokeratin no. 18 of Mall et al. [14], provided by Dr E. B. Lane, used at 1 : 25; rabbit anti-mouse
laminin antiserum (BRL 6265SA) used at 1 : 100; rat monoclonal IgM ascites antibody to stage-specific
embryonic antigen 3 (SSEA-3), provided by Dr D. Solter, used at 1 : 100; sheep antibodies to human
fibronectin (Serotec AHP 08X) used at 1 : 100; A2B5, a mouse monoclonal IgM ascites specific for
GQ ganglioside [lS], provided by Dr P. Bartlett, used at 1 : 400; and 30 H12, a mouse monoclonal IgG
antibody supematant recognizing Thy 1.2 antigen [16], provided by Dr P. Bartlett, used at 1 : 10.
Negative controls used normal rabbit or sheep serum, or monoclonal antibodies of corresponding
species and type but with irrelevant specificities.
Colonies were stained to detect alkaline phosphatase as previously described [lo].
Neutral fats were stained with Oil Red 0 (Polysciences), using the supersaturated isopropanol
method described by Lillie [ 171.
Results and Discussion
We have previously shown that a factor present in medium conditioned by ST0
fibroblasts [18] retards both the differentiation
of F9 EC cells induced with lo-’
M retinoic acid (RA) and the spontaneous differentiation
of NG2 EC cells [lo].
The present observations arose from further study of the competition between
RA and SIG-conditioned
medium.
F9 cells were seeded at clonal density (100-500 cells/75 cm*), into either 25 %
S’IDconditioned
medium, lo-’ M RA, or both together. The extent of differentiation was examined after 22 days of culture. In the absence of RA, most
colonies remained undifferentiated
as judged by cell morphology and positive
staining for alkaline phosphatase [ 191; minimal spontaneous differentiation
to
parietal endoderm-like cells was observed, as described by Sherman & Miller [4].
Colonies grown in the presence of IO-’ M RA had mostly differentiated entirely
to parietal endoderm, as identified by immunohistochemical
detection of cytokeratin no. 18 [14, 201 and laminin [21], but not SSEA-3 [20].
Colonies grown in the presence of lo-’ M RA and 25% SK&conditioned
medium contained a variety of cell morphologies.
Various differentiated cell
types were identifiable within the same colony at day 35, in addition to alkaline
phosphatase-positive
cells (fig. 1 a). Parietal endoderm cells were identified by
the above criteria (fig. 1 b, c); the presence of visceral endoderm cells was
suggested by expression of SSEA-3 [27] (fig. 1 d>. Large, refractile droplets
staining with the lipid stain Oil Red 0 [17] suggested the presence of adipocytes
(fig. 1 e). Cells with refractile bodies and processes extending up to several
millimetres were present; these bound the antibody A2B5 [14], suggesting that
they were neurones (fig. lfl. Some cells were found to express Thy-l (fig. 1 g), an
antigen present on various cell types including thymocytes 1231 and fibroblasts
1241, but not EC or parietal or visceral endoderm [25]. Large, multinucleated
cells
Fig. I. Markers of different cell types in F!9 colonies at day 35. Colonies were grown in 25 % S’IDconditioned medium and lo-’ M RA, and stained to reveal (a) cells expressing alkaline phosphatase;
(b) parietal endoderm cells expressing cytokeratin; (c) laminin; (d) cells expressing SSEA3, a marker
of visceral endoderm; (e) cells with large droplets of neutral fat; (t) neurone-like cells binding
monoclonal antibody AZBS; (g) cells expressing Thy- 1; (h) tibronectin. Brightfield micrography. Bar,
100 urn.
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570 Short notes
expressing fibronectin in their cytoplasm were observed (fig. 1 h); we were unable
to determine whether these were trophoblast cells [26] or simply degenerating
cells. Extracellular
fibronectin was also present (fig. 1 h). Several other cell
morphologies were seen which could not be identified using markers. Similar
observations were made in two separate series of experiments.
The inoculum for these cultures contained 88-92 % (n, 156-322) single cells, the
remainder being doublets. In a separate experiment, single F9 cells were placed
with finely drawn Pasteur pipettes into individual 2 cm*-culture dishes containing
25% SIG-conditioned
medium and 10e7 M RA. Of 44 colonies arising, ten
contained at least three of the cell types recognizable by morphology (parietal
endoderm, neurones, and adipocytes). Clonal expansion of differentiated
or
determined cells present in groups in the inoculum can therefore not account for
our observations.
It is possible that generation of a number of different cell lineages in the
colonies is mediated by time- or position-dependent
cues acting on residual stem
ceils, as is thought to occur in the embryo. Whilst differentiation of F9 cells relies
on the presence of an inducer, the commonly used treatment of lo-’ M RA
appears to induce so strongly that most stem cells form parietal endoderm before
other cell types can be generated. The function of SIG-conditioned
medium in
our experiments may have been to retard the effect of RA sufficiently to promote
the correct balance between stimulation of differentiation and retention of a stem
cell population.
We tested this hypothesis by subjecting F9 cells to two other treatments
expected to have a similar effect. We have previously shown, using NG2 EC
cells, that delaying the addition of lop7 M RA until day 4 of clonal growth
resulted in retention of stem cells, rather than the complete differentiation
to
parietal endoderm seen when RA is present from day 0 [271. Further, RA can be
used at 10V8 M to induce differentiation
less efficiently than at 10e7 M [5, 281.
Both these strategies were applied to clonal-density
F9 colonies, and in both
cases, cells with the properties of parietal and visceral endoderm, adipocytes,
and neurones, as well as Thy-l-positive
cells and tibronectin-positive
cells were
detected as above, after 35 days of growth. These observations are consistent
with our hypothesis. Neither treatment appeared to be as efficient as 25% STGconditioned medium/10e7 M RA in inducing multiple differentiation.
Previous studies have described conditions for the culture of F9 cells which
yield parietal [5] or visceral [6] endoderm, but not both together. Other studies
have reported neurone-like cells in bulk cultures of F9 treated with RA and (dbCAMP) [7], or when RA-db-CAMP treatment was followed by nerve growth factor
(NGF) [8]. Our results indicate that added NGF or db-CAMP is not necessary for
neurone formation. Formation of lipid cells in RA-treated F9 cultures has also
been mentioned [9]. Despite these reports, F9 is still usually referred to as
nullipotent. We have shown that single F9 cells can reproducibly yield multiple
cell types in the same colony and must therefore be considered pluripotent in
Exp Cell Res 168 (1987)
Short notes
571
vitro. It is possible that other ‘nullipotent’
EC lines may be able to differentiate in
a complex manner in vitro, given the correct conditions, and that the varied
behaviour amongst EC cell lines is attributable to regulation of, rather than
potential for, differentiation.
We thank Drs Birgitte Lane, Perry Bartlett and Davor Solter for the gifts of monoclonal antibodies.
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Received July 25, 1986
Revised version received October 13. 1986
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