Cell Differentiation, 15 (1984) 195-202 195 Elsevier Scientific Publishers Ireland, Ltd.

CDF 00281

Injection of murine embryonal carcinoma cells and embryo-derived pluripotential cells into mouse blastocysts *

K. Becker, A . M . W o b u s , U. Co n rad and J. Sch6neich

Zentralinstitut ff~r Genetik und Kulturpflanzenforschung, Akademie der Wissenschaften der DDR, GDR -4325 Gaterslehen, G.D.R.

(Received 22 October 1984)

Two pluripotential mouse cell lines, the OTT 6050-derived cell line TCE and the embryo-derived stem cell line BLC-1, were injected into blastocysts to analyze their developmental potential. The contribution of TCE cells to the embryo was found to be limited and sporadic. There was no indication of a preferential colonization of extraembryonai membranes or developmentally related tissues in adult chimeras. BLC-1 cells failed to colonize the embryo. This indicates that a normal karyotype, pluripotency, and cell surface markers which are shared by cells of early embryos are not necessarily sufficient markers for their ability to participate in embryogenesis.

mouse; teratocarcinoma; embryo; stem cell lines; blastocyst injection; chimeras

Introduction

Murine embryonal carcinoma cells have been widely used to analyze several aspects of cellular diversification processes by means of immunologi- cal, biochemical and genetic approaches. They were proposed as a model system for the study of embryonal differentiation in mammals (for review see Graham, 1977, and Martin, 1978, 1980). The most striking similarities between embryonal carcinoma (EC) cells and embryonal stem cells were demonstrated by the participation and nor- mal differentiation of EC cells in mouse embryo- genesis following blastocyst injection. This has been shown for cells from in vivo passaged teratocarcinomas (Brinster, 1974; Mintz and Ill-

" An essential part of these experiments was performed (by K.B.I at the Sir William Dunn School of Pathology, University of Oxford (U.K.).

mensee, 1975; Illmensee, 1978) as well as in vitro cultivated EC cell lines (Papaioannou et al., 1975, 1978; Stewart and Mintz, 1981; Rossant and Mc- Burney, 1982). However, it has also been shown that different cell lines revealed different proper- ties when transferred into blastocysts or aggre- gated with cleavage stage embryos. EC-derived cells did not always colonize the embryo, produced only low numbers of chimeras, revealed a limited and sporadic tissue contribution in these chimeras, interfered with normal embryogenesis, or showed retention of malignancy (for review see Papaioan- nou, 1979, and Papaioannou and Rossant, 1983).

The experimental production of chimeras by incorporation of EC cells into early mouse em- bryos is, therefore, a valuable system to analyze the relationships and interactions between EC cells and embryonic stem cells, the control of cell pro- liferation, differentiation and malignancy. More- over, EC cells were also suggested to be a vehicle

0 0 4 5 - 6 0 3 9 / 8 4 / $ 0 3 . 0 0 , 1984 Elsevier Scientific Publishers Ireland, Ltd.

196

for introducing mutant genes into the mouse germ line and thereby for creating models for human genetic diseases or for the introduction of foreign genes into mice. As far as the latter aspects are concerned, EC cells have not yet fulfilled these expectations because of the low number of germ line chimeras obtained from EC cell lines (Dewey et al., 1977; for review see Papaioannou and Ros- sant, 1983). More recently embryo-derived pluripotential cell lines were established (Evans and Kaufman, 1981) that are capable of forming a high percentage of germ line chimerism (Bradley et al., 1984).

We report here on the developmental potential of two pluripotential mouse cell lines established in our laboratory, the OTT 6050-derived EC cell line TCE (Wobus et al., submitted) and the blastocyst-derived cell line ESC-BLC-1 (Wobus et al., 1984), by injecting them into mouse blasto- cysts.

Materials and Methods

Cell lines

Embrvonal carcinoma cells The EC cell line TCE was derived from em-

bryoid bodies of teratocarcinoma OTT 6050 (ob- tained from Dr. L.C. Stevens). Feeder-indepen- dent TCE cells were found to be pluripotential when injected (s.c.) into syngeneic hosts, possess an abnormal karyotype (39, XO; two rearrange- ments, one deletion), and express cell surface anti- gens recognized by M 1/22.25, ~x-SSEA-1 and ECMA-7 monoclonal antibodies (Wobus et al., in preparation). TCE cells were cultivated in alpha- medium + 10% FCS (exp. A + a) and DMEM + 10% FCS (exp. B), respectively. Subconfluent TCE cells were treated with EGTA (0,5 mM in PBS, Stewart, 1982) or trypsin (0.025%) Tris EDTA buffer (Evans, 1972), scraped off with a rubber policeman and dispersed by pipetting in medium. The cell suspension was transferred to a plastic bacteriological culture dish and incubated for 1-3 h.

Emb(vo-derived stem cells ESC-BLC-1, a feeder-dependent pluripotential

stem cell line was derived from delayed mouse blastocysts of strain 129/ter Sv (Wobus et al., 1984). Stem cells were cultivated in DMEM + 10% FCS on X-irradiated feeder layer of mouse em- bryonal fibroblasts. Small aggregates of BLC-1 cells were isolated by t rypsin-Tr is-EDTA treat- ment of subconfluent cultures as described above.

Blasto~yst injection

Host blastocysts were flushed from uteri of random-bred PO-Gpil bb females (exp. A + a) and Gat :NMRI-Gpi l bb females (exp. B + D) on the afternoon of day 4 after natural mating, using PB-1 medium (Whittingham, 1974) plus 10% FCS. The blastocysts and small aggregates of TCE or BLC-1 cells were transferred to micro-drops of PB-1 + 10% FCS in manipulation chambers filled with liquid paraffin. Injections of cell aggregates to the blastocoelic cavities were carried out essen- tially as described by Gardner (1978), using a Leitz micromanipulator. Injected blastocysts were allowed to reexpand for 1-3 h at 37°C and were transferred to the left uterine horns of day-3 pseu- dopregnant foster mothers (exp. A + a: PO/aa; exp. B + D: C3B6F1).

Analysis

In experiments a and B, foster mothers were ~-13~ days p.c.) and killed at mid-gestation (111 1

the isolated conceptuses were dissected into the constituents of extraembryonal membranes and the fetus (for details, see Papaioannou and West, 1981). In experiment A, manipulated blastocysts were allowed to develop to term. Offspring were scored for coat and eye pigmentation and dis- sected into different tissues and organs. All tissue samples were used for analysis of GPI-1 allozymes (see West and Green, 1983) by starch gel electro- phoresis using overlaying nitrocellulose filters for staining or by cellulose acetate electrophoresis using Titan III plates (Helena Laboratories) and 'Cellogel' strips (Serva; McLaren and Buehr, 1981). For some samples the relative proportions of GPI-1 allozymes were quantified by scanning densito- metry at 550 nm with a Gelman DCD-16 comput- ing densitometer (West and Green, 1983). Eyes

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T A B L E II1

Analysis of ch imer ism at te rm following injection of T C E and BLC-1 cells into blastocysts

Exper iment Blastocysts Blastocysts Total re- Grav id re- N e w b o r n s

injected t ransplanted cipients cipients (%)

(blastocysts)

Live Newborns Chimeras

newborns examined (%)

A 277 ~ 97 12 7 32 30 26 4

(TCE) (52) (61.5) (13.3)

D 240 145 20 10 30 27 28 0

(BLC-1) (73) (41.0)

Exper iments A + a in toto.

were fixed in Bouin and analyzed histologically for melanin-producing pigment epithelium cells.

Results

As shown in Table I, 489 blastocysts were in- jected with TCE cells. In experiments a and B, 37 recipient mothers containing 261 transferred blastocysts were killed at mid-gestation. Twenty- one recipients were found to be gravid (corre- sponds to 157 transferred blastocysts). Nine out of 75 conceptuses (12%) analyzed for GPI markers revealed chimerism in one or more tissues. The pattern of TCE colonization (Table II) indicates that TCE cells did not contribute preferentially to certain tissues but seemed to colonize ex- traembryonic membranes sporadically. The extent of contribution to chimeric tissues was relatively low, as shown by GPI allozyme quantitation of the visceral yolk sac endoderm in conceptus a-20 (Fig. 1A) and by small patches of pigmented cells in the retina epithelium of a-14 and B-5 (Fig. 1B, C). It should be taken into account that quantitation of the relative proportion of GPI-1 AA in the parietal endoderm samples of chimeras a-12 and a-14 could be slightly overestimated because the possibility cannot be completely excluded that some maternal cells (Gpi-1 ~a) contaminated the parietal endo- derm in these conceptuses. Developmenta l abnormalities were found in four mid-gestation conceptuses. Extraembryonal membranes as well as the fetus were found to be very small or could not be detected at all; the placenta was highly disorganized. Three of these conceptuses showed a

contribution of TCE cells. The remaining 97 manipulated blastocysts were

transplanted to 12 foster mothers and allowed to develop to term (experiment A, Table III). Seven recipients containing 52 injected blastocysts be- came pregnant. Four of 26 newborns examined turned out to be chimeras. In three mice chimer- ism was confined to a single but different organ

T A B L E IV

Contr ibut ion of EC cell progeny to tissues of chimeric mice

der ived from blastocysts injected with T C E cells (analysis at

te rm)

Organ Newb o rn no.

(GP1) A-8 A-15 A-16 A-35

Brain - + - -

( f o r e b r a i n )

+ (cerebel lum) 61.19

Skin . . . .

Liver . . . .

Spleen . . . .

S tomach + intestine - - - +

Pancreas . . . .

G o n a d s + reprod, tract . . . .

K idney . . . . + +

Bladder - - 31.22 27.07

Lungs . . . .

Hea r t . . . .

T h y m u s . . . .

Salivary glands + - - -

Skeletal muscle . . . .

Carcass (residue) . . . .

Eyes (pigment) n.t. + - -

n.t. = not tested.

199

A -20 , y.s, endoderm )

GPI-1B GPI-1A 73.1 20.4

Propor t ion o f G P l - l a l l o z y m e s (%)

Fig. 1. T C E cell c o n t r i b u t i o n to m id -ges t a t i on concep tuses . (A) Q u a n t i t a t i o n of GPI-1 a l lozymes in the yolk sac e n d o d e r m of

c o n c e p t u s a-20. (B) Fe tus B-5, no te the p i g m e n t e d a rea ( a r row) in the re t inal ep i the l ium. (C) Fe tus a-14, h is to logica l sec t ion of the eye

s h o w i n g a small p a t c h of p i g m e n t e d re t inal ep i the l ium cells (a r row) .

A

4- a

:: :: :: ::!: : :: : ::!: : :::i!!: if: :i: :ii:: :!i i: !:: ::i:! i!:i:! ii(ill :!!ii~ii iiiii:!:(:ill iii(:ji:i i if(!!)! :i!i:(: : : : : / ~il O ~ ~ Q ~O~!! '~̧ ~i !̧ i i !~

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C -15 neuroepithelioma

GPI-1B GPI-1A 38.8 61.2

Proportion of GPI-1 allozymes (%)

Fig. 2. T C E cell c o n t r i b u t i o n to c h i m e r a A-15. (A) S ta rch gel e l ec t rophore t i c s e p a r a t i o n o f GPI -1 a l lozymes of t issue ext rac ts : a = 1 : 1 mix A + B: b = t h y m u s ; c = hear t ; d = lung; e = b l a d d e r ; f = k idney ; g = g o n a d s a n d r e p r o d u c t i v e t rac t ; h = s tomach , in tes t ine a n d

p a n c r e a s ; i = spleen; k = liver; 1 = ad ipose tissue; m = skin; n = fo reb ra in ; o = res idue of head ; p - ce rebe l lum ( tumor ) : q = res idue

of ca rcass ; r = skeletal muscle ; s - sa l ivary g land . (B) Eye showing p i g m e n t e d a reas in the c h o r o i d (ar row) . (C) Q u a n t i t a t i o n of GPI-1

a l lozymes of the cerebe l la r tumor . (D) His to log ica l sect ion of the p r imi t ive n e u r o e c t o d e r m a l ce rebe l la r t u m o r (neuroep i the l ioma) , note

the medul loep i the l ia l roset te .

200

(salivary glands, bladder, stomach plus intestine). Chimera A-15 revealed contribution of TCE cells to three tissues (Table IV). This mouse died 11 days post partum due to a cerebellar tumor. After histological examination the tumor was diagnosed to be a primitive neuroectodermal tumor (neuroep- ithelioma) containing medulloepithelial rosettes (Fig. 1D) and ependymoplastic areas. Part of the tumor was used for electrophoretic separation of GPI-1 allozymes (Fig. 2A). As demonstrated by quantitative analysis of allozyme proportions (Fig. 1C), the tumor was largely composed of TCE cell progeny (61.19%). In this animal normal differen- tiating TCE cells also colonized the bladder and the eye (Fig. 1B).

After injection of embryo-derived stem cells BLC-1 (experiment D) into 240 blastocysts we obtained 30 newborns. Twenty-eight of these were examined for GPI-1 markers by Cellogel electro- phoresis. There was no indication of chimerism in any of the samples examined (Table III).

Discussion

The purpose of the blastocyst injection experi- ments performed was to analyze the fate and developmental potential during embryogenesis of two pluripotential cell lines, a teratocarcinoma-de- rived and an embryo-derived stem cell line.

The EC cell line TCE was found able to survive and to proliferate in in vitro cultivated blastocysts (Becket, unpublished observations), to colonize the developing embryo, and to participate in the for- mation of different tissues. The frequency of chim- erism, however, was relatively low and the coloni- zation of different organs and tissues by TCE cells was very sporadic. Moreover, the level of contribu- tion to most of the chimeric tissues was also rather low. The limited and sporadic colonization pattern is in accordance with the behavior of most in vitro cultivated EC cell lines used for blastocyst injec- tion or aggregation with cleavage stage embryos (for review see Papaioannou, 1979; Papaioannou and Rossant, 1983). More recently two cell lines (METT-1, Stewart and Mintz, 1981; P10, Rossant and McBurney, 1982) were isolated, contributing to a greater extent to embryogenesis following

blastocyst injection or aggregation (Papaioannou et al., 1984).

There was no indication of a preferential col- onization to either extraembryonal membranes or developmentally related tissues in adult chimeras. Only one EC cell line (C 145b) thus far reported showed restriction in its developmental potential, which seems to be restricted to participation in the formation of the yolk sac (Papaioannou et al., 1979).

Chimera A-15 was found to have a primitive neuroectodermal tumor in the cerebellum, consist- ing largely of TCE-derived cells as demonstrated by quantitation of GPI-1 allozymes. The occur- rence of tumor-bearing chimeric mice following the transfer of EC cells into blastocysts is a widespread but infrequent phenomenon. Most of the EC cell lines that were shown to be capable of participating in embryogenesis also produced chimeras bearing tumors of EC cell origin, regard- less of their karyotypic normality or abnormality (see Papaioannou and Rossant, 1983). It is sug- gested that in these cases EC cells retained their malignant phenotype due to the failure of some EC cells to respond to embryonic signals capable of regulating EC cell proliferation and differentia- tion (Papaioannou and Rossant, 1983). As indi- cated by chimerism in two other morphologically normal tissues (bladder and eye) of mouse A-15, some of the injected TCE cells also behave like normally differentiating cells in the embryo.

Although the reasons for this colonization pat- tern are not yet understood, one could argue that these results reflect heterogeneities within the TCE cell population, which spontaneously segregates a low percentage of differentiated cell types during in vitro culture. Therefore, late integration of cells partially restricted in their potentials and delay in proliferation might account for their sporadic and limited contribution to embryogenesis. EC cell het- erogeneity is, however, not necessarily the cause of different behavior of EC cells in blastocysts as demonstrated by the formation of malignant and normal cell progeny after single cell injections with P-19 EC cells (Rossant and McBurney, 1982; Papaioannou and Rossant, 1983).

The failure of blastocyst-derived stem cells (BLC-1) to contribute to embryogenesis after

blastocyst injection shows that a normal karyo-

type, feeder-dependency and plur ipotency as dem-

onstrated by s.c. t ransplanta t ion (Wobus et al.,

1984) as well as the presence of cell surface markers

recognized by the monoclonal ant ibodies c~-SSEA-

1, M1 /22 .25 and E C M A - 7 (Walter et al., 1984)

are not necessarily sufficient markers for their

ability to par t ic ipate in embryonal development . It

is also known that in p lur ipotent EC cell lines a

normal karyotype is not a sufficient prerequisi te

for their ability to colonize the embryo (Papaioan-

nou et al., 1979; Papaioannou, 1979). Our findings

are, however, in contrast to recent results obta ined

by Evans et al. (1983) and Bradley et al. (1984).

They could demonst ra te that several embryo-de-

rived plur ipotent ia l stem cell lines are capable of

par t ic ipat ing to a large extent in embryogenesis

and germ line formation.

Because BLC-1 cells different ia te spontaneously

in vitro, this failure might reflect an immedia te

different ia t ion of the cells t ransferred to the

blastocyst that either prevents the colonizat ion of

the embryo or leads to the preferential coloniza-

tion of ex t raembryonic tissues. Alternatively, the

possibility exists that BLC-1 cells are unable to

survive inside the blastocyst. Prel iminary results

obta ined from blastocyst outgrowth exper iments

in vitro following BLC-1 injection might indicate

that immedia te different ia t ion under inappropr ia te

temporal and spatial condi t ions prevents coloniza-

tion of embryonic tissues and survival of these

cells.

Acknowledgements

Part of this work was supported by the ex-

change agreement between the Academy of Scien-

ces of the G.D.R. and the Royal Society. One of

the authors (K.B.) is part icularly indebted to Prof.

Richard L. Gardner , Dr, Chris F. G r a h a m and

John Smith for generously supplying laboratory

facilities and support ing this work. We also wish

to thank Mrs. Christa Fricke for skillful technical

assistance, Miss Judy Green and Dr. John D. West

for advice in enzyme assays, and Mrs. Maryl in

Carey and Dr. H. Ohle for histological tissue sections.

201

References

Bradley, A., M. Evans, M.H. Kaufman and E. Robertson: Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature (London) 309, 255-256 (1984).

Brinster, R.L.: The effect of cells transferred into the mouse blastocyst on subsequent development, J. Exp. Med. 140, 1049-1056 (1974).

Dewey, M.J., D.W. Martin, G.R. Martin and B. Mintz: Mosaic mice with teratocarcinoma-derived mutant cells defined in hypoxanthine phosphoribosyl-transferase. Proc. Natl. Acad. Sci. USA 74, 5564-5568 (1977).

Evans, MJ.: The isolation and properties of a clonal tissue culture strain of pluripotent mouse teratoma cells. J. Em- bryol. Exp. Morphol. 28, 163-176 (1972).

Evans, M.J. and M.H. Kaufman: Establishment in culture of pluripotential cells from mouse embryos. Nature (London) 292, 154-156 (1981).

Evans, M.J, E. Robertson, A. Bradley and M.H. Kaufman: The relationship between embryonal carcinoma cells and embryos. In: Current Problems in Germ Cell Differentia- tion, Br. Soc. Dev. Biol. Syrup. 7. ed. A. McLaren and C.C. Wylie (Cambridge University Press, Cambridge) pp. 139 155 (1983).

Gardner, R.L.: Production of chimaeras by injecting cells or tissue into the blastocyst. In: Methods in Mammalian Re- production, ed. J.C. Daniel (Academic Press, New York) pp. 137-165 (1978).

Graham, C.F.: Teratocarcinoma cells and normal mouse em- bryogenesis. In: Concepts in Mammalian Embryogenesis, ed. M.I. Sherman (MIT Press, Cambridge, MA) pp. 315-394 (1977).

Illmensee, K.: Reversion of malignancy and normalized differ- entiation of teratocarcinoma cells in chimaeric mice. In: Genetic Mosaics and Chimaeras in Mammals, ed. L.B. Russel (Plenum Press, New York) pp. 3 25 (1978).

Martin, G.R.: Advantages and limitations of teratocarcinoma stem cells as models of development. In: Development in Mammals, Vol. 3, ed. M.H. Johnson (Elsevier/North Hol- land Biomedical Press, Amsterdam)pp. 225-265 (1978).

Martin, G.R.: Teratocarcinomas and mammalian embryogene- sis. Science 209, 768-776 (1980).

McLaren, A. and M. Buehr: GPI expression in female germ cells of the mouse. Genet. Res. 37, 303 309 (1981).

Mintz, B. and K. lllmensee: Normal genetically mosaic mice produced from malignant teratocarcinoma cells. Proc. Natl. Acad. Sci. USA 72, 3585-3589 (1975),

Papaioannou, V.E.: Interactions between mouse embryos and teratocarcinomas. In: Cell Lineage, Stem Cells and Cell Differentiation, INSERM Symposium No. 10, ed. N. Le Douarin (Elsevier/North Holland Biomedical Press, Amsterdam) pp. 141-155 (1979).

Papaioannou, V.E. and J. Rossant: Effects of the embryonic environment on proliferation and differentiation of em- bryonal carcinoma cells. Cancer Surv. 2, 165-183 (1983).

202

Papaioannou, V.E. and J.D. West: Relationship between the parental origin of the X chromosomes, embryonic cell lin- eage and X chromosome expression in mice. Genet. Res. 37, 183-197 (1981).

Papaioannou, V.E., E.P. Evans, R.L. Gardner and C.F. Grahm: Growth and differentiation of an embryonal carcinoma cell line (C145b). J. Embryol. Exp. Morphol. 54, 277-295 (1979).

Papaioannou, V.E., R.L. Gardner, M.W. McBurney, C. Babinet and M.J. Evans: Participation of cultured teratocarcinoma cells in mouse embryogenesis. J. Embryol. Exp. Morphol. 44, 93-104 (1978).

Papaioannou, V.E., M.W. McBurney, R.L. Gardner and M.J. Evans: Fate of teratocarcinoma cells injected into early mouse embryos. Nature (London) 258, 70-73 (1975).

Papaioannou, V.E., B.K. Waters and J. Rossant: Interactions of embryonal carcinoma cells and normal embryonat cells. Cell Differ. 15, 175-179 (1984).

Rossant, J. and M.W. McBurney: The developmental potential of a euploid male teratocarcinoma cell line after blastocyst injection. J. Embryol. Exp. Morphol. 70, 99 112 (1982).

Stewart, C.L.: Formation of viable chimaeras by aggregation between teratocarcinomas and preimplantation mouse em- bryos. J. Embryol. Exp. Morphol. 67, 167-179 (1982).

Stewart, T.A. and B. Mintz: Successive generations of mice produced from an established culture line of euploid teratocarcinoma cells. Proc. Natl. Acad. Sci. USA 78, 6314-6318 (1981).

Walter, G., A. Intek, A.M. Wobus and J. Sch6neich: Serologi- cal characterization of pluripotent mouse embryonal stem cell lines and transformed derivatives. Cell Differ. 15, 147-151 (1984).

West, J.D. and J.F. Green: The transition from oocyte-coded to embryo-coded glucose phosphate isomerase in the early mouse embryo. J. Embryol. Exp. Morphol. 78, 127-140 (1983).

Whittingham, D.G.: Embryo banks in the future of develop- mental genetics. Genetics 78, 395-402 (1974).

Wobus, A.M., H. Holzhausen, C. Block, A. lntek, K. Becker, G. Walter, J. Forejt and J. Sch6neich: Establishment and characterization of the pluripotent mouse teratocarcinoma cell line TCE. Submitted.

Wobus, A.M., H. Holzhausen, P. J~_kel and J'. Sch6neich: Characterization of a pluripotent stem cell line derived from a mouse embryo. Exp. Cell Res. 152, 212-219 (1984).