Presence of multipotential hemopoietic cell ins ...avenue de la Belle Gabrielle, F- 94130 Nogent sur Marne, France. 2 Author's address: Cancer Research Unit, The Walter and Eliza Hall

J. Embryol. exp. Morph. Vol. 61, pp. 51-59, 1981Printed in Great Britain © Company of Biologists Limited 1981

Presence of multipotentialhemopoietic cells in teratocarcinoma

cultures

By CLAUDE A. CUDENNEC1 AND GREGORY R. JOHNSON2

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 todifferentiate in vitro to produce red blood cells of the primitive hemopoietic cell popu-lation of the mouse embryo. The present paper demonstrates the presence in the samecultures of multipotential cells capable of giving rise to colonies containing erythrocytes,macrophages, and megakaryocytes when stimulated by pokeweed-mitogen spleen-cell condi-tioned medium.

INTRODUCTION

Teratocarcinoma cells have been shown to be able to differentiate alonghemopoietic lineages either in vitro (Cudennec & Nicolas, 1977) (see alsoGraham, 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 formationregularly takes place in organ cultures of the clonal teratocarcinoma linePCC3/A/1 (Jakob et al. 1973; Nicolas et al. 1976). Morphological observationof blood-cell differentiation in the cultures showed that large primitiveerythrocytes developed but non-nucleated erythroid cells never appeared underthese conditions. The erythropoietic foci were, in all cases, associated withvesicles lined by an endodermal-like epithelium. In addition, the blood islandsthat arose in the cultures were transitory structures in which erythropoiesisceased completely after 5 to 10 days.

Biochemical analysis showed that the red cells synthesize a set of embryonichemoglobins characteristic of the primitive, yolk-sac type, generation oferythrocytes (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 MedicalResearch, 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-sachematopoietic potential by teratocarcinoma cells.

Such a developmental capability is never expressed in the histiotypic tissuecultures of this teratocarcinoma line although various differentiated tissues,especially endodermal epithelium, have been shown to arise under theseconditions (Nicolas et al. 1975). All the attempts we have made to detecthemoglobin synthesis in such cultures have been unsuccessful.

To further characterize the developmental capabilities of the hemopoieticcells which differentiate in organ cultures of teratocarcinoma cells, we havedetermined the responsiveness of such cells to stimulating factors known tobe 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 ofneutrophils, macrophages, eosinophils and megakaryocytes fiom mouse bone-marrow cells (Metcalf, Parker, Chester & Kincade, 1974; Metcalf, MacDonald,Odartchenko & Sordat, 1975). In addition SCM contains (a) factor(s) capableof stimulating multipotential hemopoietic cells to form clones containingerythrocytes, neutrophils, macrophages, eosinophils and megakaryocytes(Johnson & Metcalf, 1977a). These mixed hemopoietic-colony-forming cellsoccur most frequently in yolk sac, early fetal liver and peripheral blood culturesbut 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 eitherfrom the hemopoietic foci present in organ cultures of teratocarcinoma cellsor from histiotypic cultures.

MATERIALS AND METHODS

Cultures ofPCC3/A/l cell line (see Fig. 1)

Teratocarcinoma cells were maintained undifferentiated by serial trans-plantations in 10 cm plastic Petri dishes (Nunclon). The medium (Dulbecco'smodified Eagle's medium: DMEM) was supplemented by 15 % FCS (GIBCO).To set up organ cultures, the cells of a confluent culture were detached fromthe plastic by gentle pipetting. The resultant cell suspension was spun for 10 minat 800 g and the pellet divided into 4 parts (approx. 107 cells each) which wereindividually transferred onto a Millipore filter (0-22 [im pore size). Thesecultures were kept at the gas-medium (DMEM + FCS) interphase (PrimaryOrganotypic cultures :PO cultures). Fifty percent of the PO cultures showedblood foci between day 10 and 30 of incubation (Cudennec & Nicolas,1977). After 10 days some PO cultures were subcultured in secondaryorganotypic (SO) cultures by fragmentation. Each PO culture was dividedinto 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 nodifferences in the nature and relative percentages of the various differentiatedhemopoietic cells identified by analysis of blood cell smears (Cudennec et al.1979). All cultures were performed at 37 °C in an atmosphere composed of10 % CO2 in air.

Preparation of blood-cell suspension

Tissues obtained from blood-forming areas of approximately fifty 9-day-oldSO cultures were collected and transferred into Eisen's balanced salt solution(EBSS). The aggregates were disrupted by gentle pipetting, the cells werewashed twice in EBSS and resuspended in 2 ml cold EBSS. The cells werelayered onto 10 ml EBSS containing 50 % (w/v) FCS and allowed to sedimentfor 75 min. In order to obtain a pure single cell suspension, we discarded thelower fraction of 2 ml containing cell aggregates and the upper fraction of 2 mlcontaining debris. Viability of cells was determined by Trypan blue exclusionand the cell numbers evaluated in a hemocytometer. This suspension was thesource of the cells subsequently transferred into agar culture containingpokeweed-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 cellsper ml in RPMI-1640 containing 5 % heat-inactivated human plasma and 0-05ml 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. Thesupernatant 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 weresuspended 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 for7 days.

Scoring of agar cultures

Cultures were scored for the presence of erythroid (red or pink) colonies ornon-erythroid (white) colonies using a dissection microscope at x40magnification.

The cellular composition of the colonies was determined by picking offindividual colonies, smearing them on glass slides and staining them either


PCC3/A/1cell culture

Mechanicaldissociation

Centrifugation

Cloning of cellsfrom the blood

islands

Agar culture

Fig. 1. Different types of culture used to allow erythroid capabilities to be expressedby cells of the PCC3/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 incacodylate 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 Reichert0MU2 ultramicrotome. The sections were double stained with uranyl acetateand lead citrate, and observed in a Hitachi HS9 electron microscope.

RESULTS

Undifferentiated tumor cells (Embryonal Carcinoma cells:EC cells, the stemcells of the tumor) were harvested from exponentially growing cell cultures inwhich confluency had not yet been reached. The cell suspension was plated insoft agar culture with or without spleen-conditioned medium (SCM). Afterseven days of incubation, similar results were obtained irrespective of thepresence of SCM in the cultures. Compact aggregates developed in whichdifferentiated tissues, such as rhythmically beating muscle cells could sometimesbe identified. But in no instance were hemopoietic colonies observed.

A second series of experiments was performed using cells harvested fromorgan cultures of PCC3/A/1 cell line (Fig. 1). As previously mentioned this


r

E

Fig. 2. Electron micrograph of a mixed-erythroid colony derived from terato-carcinoma cells, showing two different cell types: Erythroblast (E) and Mega-karyocyte (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 differentiatedtissues including blood islands. The spatial organization of the differentiatedtissues was chaotic. Consequently the mechanical picking-up of cells fromblood-forming areas led to heterogenous cell suspensions. These suspensionswere composed not only of hemopoietic cells but also of cells, or clumps of cells,from surrounding tissues - namely endodermal-like epithelium - as well as ECcells, the stem cells of the tumor.

In order to separate the single cells and the clumps, the suspensions weresedimented in 50 % FCS-containing culture medium. As described in the'Materials and methods' section, only the single cells were used in the clonalagar culture system.

In addition to aggregates similar to those obtained in soft agar cultures ofEC cells from histiotypic cultures, colonies of hemopoietic cells developed in7-day soft-agar cultures of cells obtained from SO cultures. Both erythroid andnon-erythroid colonies could be distinguished according to their size, colour


Table 1. Frequency of erythroid and non-erythroid colony-forming cells in agarcultures of organ-cultured PCC31A11 cells*

Hemopoietic colony-forming cells per 105 cells cultured

Batch of human plasma Erythroid Non-erythroid

1 11 ±2 N.S.1 19±5 246±352 3±1 132±203 1±1 89±124 2±1 116±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-conditionedmedium, 20 % human plasma.

and cellular composition. No hemopoietic colonies developed in controlcultures without SCM. No colonies of erythroid cells developed from sus-pensions of cells prepared from SO cultures older than 20 days in whichhemopoietic activity had ceased.

Erythroid colonies were characterized by the presence of erythroid cellseasily visualized in living cultures due to extensive hemoglobinization. Thesecells were evenly distributed in colonies composed of up to several hundredsof cells and represented 40 to 70 % of the total colony cellularity. Whenexamined by electron microscopy, the erythroid colonies contained erythroidcells at all developmental stages of hemoglobinization. In some colonies, theerythroid cells could be seen mixed with megakaryocytes (Figs. 2 and 3),macrophages and occasional neutrophils. These colonies appeared to be similarto 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 werecomposed of a tight center and a loose outer mantle of cells, the remainderbeing composed of dispersed cells.

To determine the cellular composition of the erythroid and non-erythroidhemopoietic 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 cellssmeared onto microsome slides, fixed with methanol and stained with benzidineand Giemsa stains. When examined microscopically, stained smears oferythroid colonies contained benzidine-positive erythroid cells at all stages of


•v-k^\ V.

Fig. 3. Detail of the cytoplasm of a Megakaryocyte developed in a mixed-erythroidcolony 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 containedmegakaryocytes, 30 colonies were sequentially removed from day-7 culturesand placed onto chick-egg-albumin-coated slides, allowed to air dry, followedby acetone fixation and acetylcholinesterase staining. Twenty of the 30colonies contained acetylcholinesterase-positive megakaryocyte. A similarprocedure was applied to both erythroid and non-erythroid colonies in order todetermine the overall incidence of megakaryocyte clones. A total of 96 sequentialcolonies were removed and stained with acetylcholinesterase and Giemsa stains.Eleven of the colonies contained only megakaryocytes, 21 contained mega-karyocytes and erythroid cells and the remaining 64 colonies contained onlymacrophages.

The frequency on day 7 of culture, of colonies developing from suspensionsof blood-forming SO cultured cells is reported in Table 1. Five series of cultureswere performed from five different sets of teratocarcinoma cultures, using a totalof four batches of human plasma as medium supplement. The cell density at


seeding varied from 2 x 104 to 2 x 105 cells per 1 ml culture but did not affectthe frequency of colonies in the cultures of a replicate series.

The differences in colony frequency from experiment to experiment wereprobably due to the different batches of human plasma used and the com-position of the teratocarcinoma cell suspension cultured.

Human plasma number 1 was pretested in Melbourne on normal fetal livercells before being used in teratocarcinoma cell cultures. Batch numbers2, 3 and 4 were purchased from a hospital in Paris. Mixed erythroid colonyformation obtained from teratocarcinoma cell cultures appeared to be highlydependent on the human-plasma batch (some of them were absolutelyinsufficient for the agar colony growth). The frequency of erythroid colonyprecursors obtained in cultures containing human plasma number 1 correspondsapproximately to those found in mouse embryonic yolk sac on day 12 ofgestation (Johnson & Metcalf, 1977&).

DISCUSSION AND CONCLUSION

Previous results have shown that the PCC3/A/1 teratocarcinoma line is ableto produce spontaneously in vitro red cells pertaining to the primitive, yolk-sac-type, generation (Cudennec & Nicolas, 1977; Cudennec et al. 1979). Thistransient differentiation represents the unique erythroid capability expressed byPCC3/A/1 in mass culture. The lack of definitive erythrocyte formation in sucha situation might result either from the inadequacy of the culture system toprovide the specific stimulation to the erythroid precursors or from thedepletion of hemopoietic stem-cell compartment due to the differentiation ofeach 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 containprecursors of several hemopoietic lineages. It also implies that the short lifespan of erythropoiesis in these cultures is not due to the depletion of thehemopoietic stem-cell compartment, but possibly due to the lack of appropriatestimulation for further differentiation. During normal murine development thefetal liver succeeds the yolk sac as the primary fetal hemopoietic organ. Notissues structurally similar to fetal liver have been observed in organ culturesof PCC3/A/1 cells. Consequently, failure of hepatic differentiation may resultin a lack of the necessary regulatory signals for further hemopoietic differenti-ation of precursors present in the yolk sac. This hypothesis is consistent withdata showing that fetal liver hemopoiesis is sustained by the migration of cellsinto the liver primordium at the 28-somite stage (Johnson & Moore, 1975).


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(Received 22 May 1980, revised 17 August 1980)

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Presence of multipotential hemopoietic cell ins ...avenue de la Belle Gabrielle, F- 94130 Nogent sur Marne, France. 2 Author's address: Cancer Research Unit, The Walter and Eliza Hall