J. Embryol. exp. Morph. Vol. 61, pp. 51-59, 1981
Printed in Great Britain © Company of Biologists Limited 1981
Presence of multipotential
hemopoietic cells in teratocarcinoma
cultures
By CLAUDE A. CUDENNEC 1 AND GREGORY R. JOHNSON 2
From the Institut d'Embryologie, C.N.R.S. & College de France,
Nogent sur Marne
SUMMARY
Previous investigations have shown that the teratocarcinoma line PCC3/A/1 is able to
differentiate in vitro to produce red blood cells of the primitive hemopoietic cell population of the mouse embryo. The present paper demonstrates the presence in the same
cultures of multipotential cells capable of giving rise to colonies containing erythrocytes,
macrophages, and megakaryocytes when stimulated by pokeweed-mitogen spleen-cell conditioned medium.
INTRODUCTION
Teratocarcinoma cells have been shown to be able to differentiate along
hemopoietic lineages either in vitro (Cudennec & Nicolas, 1977) (see also
Graham, 1977), or in vivo (Mintz & Illmensee, 1975; Papaioannou et al. 1975;
Illmensee & Mintz, 1976; Papaioannou et al. 1978; Cudennec & Salaiin, 1979).
As previously described (Cudennec & Nicolas, 1977) blood-island formation
regularly takes place in organ cultures of the clonal teratocarcinoma line
PCC3/A/1 (Jakob et al. 1973; Nicolas et al. 1976). Morphological observation
of blood-cell differentiation in the cultures showed that large primitive
erythrocytes developed but non-nucleated erythroid cells never appeared under
these conditions. The erythropoietic foci were, in all cases, associated with
vesicles lined by an endodermal-like epithelium. In addition, the blood islands
that arose in the cultures were transitory structures in which erythropoiesis
ceased completely after 5 to 10 days.
Biochemical analysis showed that the red cells synthesize a set of embryonic
hemoglobins characteristic of the primitive, yolk-sac type, generation of
erythrocytes (Cudennec, Delouvee & Thiery, 1979). Consequently, it appears
1
Author's address: Institut d'Embryologie, C.N.R.S.. & College de France, 49 bis,
avenue
de la Belle Gabrielle, F- 94130 Nogent sur Marne, France.
2
Author's address: Cancer Research Unit, The Walter and Eliza Hall Institute of Medical
Research, P.O. Royal Melbourne Hospital, Victoria, 3050, Australia.
52
C. A. CUDENNEC AND G. R. JOHNSON
that the blood foci formed in organ culture are the expression of a yolk-sac
hematopoietic potential by teratocarcinoma cells.
Such a developmental capability is never expressed in the histiotypic tissue
cultures of this teratocarcinoma line although various differentiated tissues,
especially endodermal epithelium, have been shown to arise under these
conditions (Nicolas et al. 1975). All the attempts we have made to detect
hemoglobin synthesis in such cultures have been unsuccessful.
To further characterize the developmental capabilities of the hemopoietic
cells which differentiate in organ cultures of teratocarcinoma cells, we have
determined the responsiveness of such cells to stimulating factors known to
be operative on hemopoietic cells of both adult and fetal origin.
Media conditioned by pokeweed-mitogen-stimulated spleen cells (SCM),
when used in soft agar cultures, stimulates the formation of colonies of
neutrophils, macrophages, eosinophils and megakaryocytes fiom mouse bonemarrow cells (Metcalf, Parker, Chester & Kincade, 1974; Metcalf, MacDonald,
Odartchenko & Sordat, 1975). In addition SCM contains (a) factor(s) capable
of stimulating multipotential hemopoietic cells to form clones containing
erythrocytes, neutrophils, macrophages, eosinophils and megakaryocytes
(Johnson & Metcalf, 1977a). These mixed hemopoietic-colony-forming cells
occur most frequently in yolk sac, early fetal liver and peripheral blood cultures
but are also present in cultures of adult spleen or bone-marrow cells (Johnson
& Metcalf, 1911a, b).
The effect of the various colony stimulating factors (Burgess, Metcalf &
Russel, 1978) present in SCM was tested in agar cultures of cells obtained either
from the hemopoietic foci present in organ cultures of teratocarcinoma cells
or from histiotypic cultures.
MATERIALS AND METHODS
Cultures ofPCC3/A/l
cell line (see Fig. 1)
Teratocarcinoma cells were maintained undifferentiated by serial transplantations in 10 cm plastic Petri dishes (Nunclon). The medium (Dulbecco's
modified Eagle's medium: DMEM) was supplemented by 15 % FCS (GIBCO).
To set up organ cultures, the cells of a confluent culture were detached from
the plastic by gentle pipetting. The resultant cell suspension was spun for 10 min
at 800 g and the pellet divided into 4 parts (approx. 107 cells each) which were
individually transferred onto a Millipore filter (0-22 [im pore size). These
cultures were kept at the gas-medium (DMEM + FCS) interphase (Primary
Organotypic cultures :PO cultures). Fifty percent of the PO cultures showed
blood foci between day 10 and 30 of incubation (Cudennec & Nicolas,
1977). After 10 days some PO cultures were subcultured in secondary
organotypic (SO) cultures by fragmentation. Each PO culture was divided
into about fifty 1 mm3 pieces which were individually transferred onto a new
Multipotential hemopoietic cells in teratocarcinoma cultures
53
Millipore filter. The percentage of SO cultures showing red-cell foci was 85 %
and hemopoietic foci occurred sooner in SO cultures than in PO cultures.
In spite of these differences between PO and SO cultures, there were no
differences in the nature and relative percentages of the various differentiated
hemopoietic cells identified by analysis of blood cell smears (Cudennec et al.
1979). All cultures were performed at 37 °C in an atmosphere composed of
10 % CO 2 in air.
Preparation of blood-cell suspension
Tissues obtained from blood-forming areas of approximately fifty 9-day-old
SO cultures were collected and transferred into Eisen's balanced salt solution
(EBSS). The aggregates were disrupted by gentle pipetting, the cells were
washed twice in EBSS and resuspended in 2 ml cold EBSS. The cells were
layered onto 10 ml EBSS containing 50 % (w/v) FCS and allowed to sediment
for 75 min. In order to obtain a pure single cell suspension, we discarded the
lower fraction of 2 ml containing cell aggregates and the upper fraction of 2 ml
containing debris. Viability of cells was determined by Trypan blue exclusion
and the cell numbers evaluated in a hemocytometer. This suspension was the
source of the cells subsequently transferred into agar culture containing
pokeweed-mitogen-stimulated spleen-conditioned medium (SCM).
Preparation of spleen-conditioned medium
C57B1 spleen cells were incubated for 5 days at a concentration of 2 x 106 cells
per ml in RPMI-1640 containing 5 % heat-inactivated human plasma and 0-05
ml of a 1:15 dilution of pokeweed mitogen per ml of culture medium (GIBCO).
After incubation, the media were centrifuged for 10 min at 3000 g. The
supernatant fluid was then harvested and Millipore filtered.
Agar cultures
Cells were cultured in agar medium as described previously (Metcalf et al.
1974; Metcalf et al. 1975; Johnson & Metcalf, \911a). Briefly, cells were
suspended in 0-3 % agar in DMEM supplemented with -20 % human plasma.
One ml suspensions were plated into plastic Petri dishes containing 0-2 ml SCM
(or 0-2 ml DMEM in control experiments). These cultures were incubated for
7 days.
Scoring of agar cultures
Cultures were scored for the presence of erythroid (red or pink) colonies or
non-erythroid (white) colonies using a dissection microscope at x40
magnification.
The cellular composition of the colonies was determined by picking off
individual colonies, smearing them on glass slides and staining them either
54
C. A. CUDENNEC AND G. R. JOHNSON
Mechanical
dissociation
PCC3/A/1
cell culture
Centrifugation
Cloning of cells
from the blood
islands
Agar culture
Fig. 1. Different types of culture used to allow erythroid capabilities to be expressed
by cells of the PCC 3 /A/1 teratocarcinoma line.
with benzidine and Giemsa or hematoxylin (McLeod, Shreeve & Axelrod, 1974)
or for acetylcholinesterase detection (Karnovsky & Roots, 1964).
Some erythroid colonies were fixed for 20 min in 3 % glutaraldehyde in
cacodylate buffer and post fixed for 1 h in 1 % OsO4. After alcohol dehydration,
the colony was embedded in Epon 812, and ultrathin sections cut with a Reichert
0MU2 ultramicrotome. The sections were double stained with uranyl acetate
and lead citrate, and observed in a Hitachi HS9 electron microscope.
RESULTS
Undifferentiated tumor cells (Embryonal Carcinoma cells:EC cells, the stem
cells of the tumor) were harvested from exponentially growing cell cultures in
which confluency had not yet been reached. The cell suspension was plated in
soft agar culture with or without spleen-conditioned medium (SCM). After
seven days of incubation, similar results were obtained irrespective of the
presence of SCM in the cultures. Compact aggregates developed in which
differentiated tissues, such as rhythmically beating muscle cells could sometimes
be identified. But in no instance were hemopoietic colonies observed.
A second series of experiments was performed using cells harvested from
organ cultures of PCC3/A/1 cell line (Fig. 1). As previously mentioned this
Multipotential hemopoietic cells in teratocarcinoma cultures
55
r
E
Fig. 2. Electron micrograph of a mixed-erythroid colony derived from teratocarcinoma cells, showing two different cell types: Erythroblast (E) and Megakaryocyte (M). (The colony was picked out of a day-7 agar culture containing SCM.)
(Scale bar = 2 fim.)
tumor maintained under such conditions gave rise to various differentiated
tissues including blood islands. The spatial organization of the differentiated
tissues was chaotic. Consequently the mechanical picking-up of cells from
blood-forming areas led to heterogenous cell suspensions. These suspensions
were composed not only of hemopoietic cells but also of cells, or clumps of cells,
from surrounding tissues - namely endodermal-like epithelium - as well as EC
cells, the stem cells of the tumor.
In order to separate the single cells and the clumps, the suspensions were
sedimented in 50 % FCS-containing culture medium. As described in the
'Materials and methods' section, only the single cells were used in the clonal
agar culture system.
In addition to aggregates similar to those obtained in soft agar cultures of
EC cells from histiotypic cultures, colonies of hemopoietic cells developed in
7-day soft-agar cultures of cells obtained from SO cultures. Both erythroid and
non-erythroid colonies could be distinguished according to their size, colour
56
C. A. CUDENNEC AND G. R. JOHNSON
Table 1. Frequency of erythroid and non-erythroid colony-forming cells in agar
cultures of organ-cultured PCC31A11 cells*
Hemopoietic colony-forming cells per 105 cells cultured
Batch of human plasma
Erythroid
Non-erythroid
1
1
2
3
4
11 ±2
19±5
3±1
1±1
2±1
N.S.
246±35
132±20
89±12
116±31
N.S., non-scored.
* Data obtained from five separate series of PCC3/A/1 organ cultures.
Mean number of colonies (± S.E.M.) determined from eight to ten replicate 1 ml agar
cultures each containing 0-2 ml pokeweed-mitogen-stimulated spleen-cell-conditioned
medium, 20 % human plasma.
and cellular composition. No hemopoietic colonies developed in control
cultures without SCM. No colonies of erythroid cells developed from suspensions of cells prepared from SO cultures older than 20 days in which
hemopoietic activity had ceased.
Erythroid colonies were characterized by the presence of erythroid cells
easily visualized in living cultures due to extensive hemoglobinization. These
cells were evenly distributed in colonies composed of up to several hundreds
of cells and represented 40 to 70 % of the total colony cellularity. When
examined by electron microscopy, the erythroid colonies contained erythroid
cells at all developmental stages of hemoglobinization. In some colonies, the
erythroid cells could be seen mixed with megakaryocytes (Figs. 2 and 3),
macrophages and occasional neutrophils. These colonies appeared to be similar
to those which develop from embryonic hemopoietic cells described previously
(Johnson & Metcalf, 1977a, b).
Non-erythroid hemopoietic colonies were numerous in agar cultures (Table 1).
They appeared most commonly unicentric but polymorphic. Some were
composed of a tight center and a loose outer mantle of cells, the remainder
being composed of dispersed cells.
To determine the cellular composition of the erythroid and non-erythroid
hemopoietic colonies, individual colonies were stained with either benzidine,
Giemsa or acetylcholinesterase stains. Thus, after seven days of culture,
individual red-coloured colonies were sequentially removed and the cells
smeared onto microsome slides, fixed with methanol and stained with benzidine
and Giemsa stains. When examined microscopically, stained smears of
erythroid colonies contained benzidine-positive erythroid cells at all stages of
Multipotential hemopoietic cells in teratocarcinoma cultures
•v-k^\
57
V.
Fig. 3. Detail of the cytoplasm of a Megakaryocyte developed in a mixed-erythroid
colony derived from teratocarcinoma cells. Note the presence of typical a granules.
(Scale bar = 0-5 fim.)
maturation, intermingled with macrophages, megakaryocytes and occasionally,
neutrophils. To determine the proportion of erythroid colonies that contained
megakaryocytes, 30 colonies were sequentially removed from day-7 cultures
and placed onto chick-egg-albumin-coated slides, allowed to air dry, followed
by acetone fixation and acetylcholinesterase staining. Twenty of the 30
colonies contained acetylcholinesterase-positive megakaryocyte. A similar
procedure was applied to both erythroid and non-erythroid colonies in order to
determine the overall incidence of megakaryocyte clones. A total of 96 sequential
colonies were removed and stained with acetylcholinesterase and Giemsa stains.
Eleven of the colonies contained only megakaryocytes, 21 contained megakaryocytes and erythroid cells and the remaining 64 colonies contained only
macrophages.
The frequency on day 7 of culture, of colonies developing from suspensions
of blood-forming SO cultured cells is reported in Table 1. Five series of cultures
were performed from five different sets of teratocarcinoma cultures, using a total
of four batches of human plasma as medium supplement. The cell density at
58
C. A. CUDENNEC AND G. R. JOHNSON
seeding varied from 2 x 104 to 2 x 105 cells per 1 ml culture but did not affect
the frequency of colonies in the cultures of a replicate series.
The differences in colony frequency from experiment to experiment were
probably due to the different batches of human plasma used and the composition of the teratocarcinoma cell suspension cultured.
Human plasma number 1 was pretested in Melbourne on normal fetal liver
cells before being used in teratocarcinoma cell cultures. Batch numbers
2, 3 and 4 were purchased from a hospital in Paris. Mixed erythroid colony
formation obtained from teratocarcinoma cell cultures appeared to be highly
dependent on the human-plasma batch (some of them were absolutely
insufficient for the agar colony growth). The frequency of erythroid colony
precursors obtained in cultures containing human plasma number 1 corresponds
approximately to those found in mouse embryonic yolk sac on day 12 of
gestation (Johnson & Metcalf, 1977&).
DISCUSSION AND CONCLUSION
Previous results have shown that the PCC3/A/1 teratocarcinoma line is able
to produce spontaneously in vitro red cells pertaining to the primitive, yolk-sactype, generation (Cudennec & Nicolas, 1977; Cudennec et al. 1979). This
transient differentiation represents the unique erythroid capability expressed by
PCC3/A/1 in mass culture. The lack of definitive erythrocyte formation in such
a situation might result either from the inadequacy of the culture system to
provide the specific stimulation to the erythroid precursors or from the
depletion of hemopoietic stem-cell compartment due to the differentiation of
each stem cell toward the primitive hemopoietic generation.
Present results show that, in addition to well-differentiated hemopoietic cells,
blood islands differentiating in organ culture of teratocarcinoma cells contain
precursors of several hemopoietic lineages. It also implies that the short life
span of erythropoiesis in these cultures is not due to the depletion of the
hemopoietic stem-cell compartment, but possibly due to the lack of appropriate
stimulation for further differentiation. During normal murine development the
fetal liver succeeds the yolk sac as the primary fetal hemopoietic organ. No
tissues structurally similar to fetal liver have been observed in organ cultures
of PCC3/A/1 cells. Consequently, failure of hepatic differentiation may result
in a lack of the necessary regulatory signals for further hemopoietic differentiation of precursors present in the yolk sac. This hypothesis is consistent with
data showing that fetal liver hemopoiesis is sustained by the migration of cells
into the liver primordium at the 28-somite stage (Johnson & Moore, 1975).
Multipotential hemopoietic cells in teratocarcinoma cultures
59
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