Proc. Nat. Acad. Sci. USA Vol. 73, No. 2, pp. 549-553, February 1976 Cell Biology Totipotency and normal differentiation of single teratocarcinoma cells cloned by injection into blastocysts (mouse teratoma/embryonal carcinoma/embryoid body core cells/allophenic mice/genetic mosaicism) KARL ILLMENSEE AND BEATRICE MINTZ* Institute for Cancer Research, Fox Chase, Philadelphia, Pennsylvania 19111 Contributed by Beatrice Mintz, November 19,1975 ABSTRACT A definitive test for developmental totipo- tency of mouse malignant teratocarcinoma cells was con- ducted by cloning singly injected cells in genetically marked blastocysts. Totipotency was conclusively shown in an adult mosaic female whose tumor-strain cells had made substantial contributions to all of the wide range of its somatic tissues analyzed; the clonally propagated cell lineage had therefore differentiated in numerous normal directions. The test cells were from "cores" of embryoid bodies of a euploid, chromo- somally male (X/Y), ascites tumor grown only in vivo by transplantation for 8 years. The capacity of cells from the same source to differentiate, in a phenotypic male, into rep- roductively functional sperms, has been shown in our previ- ous experiments [(1975) Proc. Nat. Acad. Sci. USA 72, 3585- 3589]. Cells from this transplant line therefore provide mate- rial suitable for projected somatic and germ-line genetic analyses of mammalian differentiation based on "cycling" of mutation-carrying tumor cells through developing embryos. In some animals obtained from single-cell injections, tumor- derived cells were sporadically distributed in developmental- ly unrelated tissues. These cases can be accounted for by de- layed and haphazard cellular integration, and by a marked degree of sustained cellular developmental flexibility in early mammalian development, irrespective of certain classi- cal "germ-layer" designations. All mosaic mice obtained have thus far been free of teratomas. In one case, the inject- ed stem cell contributed only to the pancreas and gave rise to a malignancy resembling pancreatic adenocarcinoma. The high modal frequency of euploidy in these individually test- ed cells thus tends to indicate that a near-normal chromo- some complement is sufficient for total restoration of orderly gene expression in a normal embryonic environment; it may also be necessary for teratoma stem-cell proliferation to be terminated there. Teratomas, unlike other tumors, often comprise a variety of tissues, derived from teratocarcinoma, or embryonal carci- noma, stem cells (1, 2). Pluripotency of the stem cells was demonstrated when solid tumors of varied tissue composi- tion were obtained from transfers of single cells to graft hosts (3). However, the consistent absence of certain tissues (4), and the immaturity and aberrations of others, in terato- mas has until recently left open the question whether em- bryonal carcinoma cells are in fact developmentally totipo- tent. Full realization of normal developmental potentialities would be expected to occur, for embryonal carcinoma as for normal embryonal cells, only in the normally organized en- vironment of an early embryo. We therefore undertook ex- periments in which mouse teratocarcinoma cells were inject- ed into the cavity of genetically marked blastocysts (5, 6). The donor cells were from the "cores" of teratoma embryoid bodies grown only in vvo as a transplantable ascites since the tumor (OTT 6050) was isolated in 1967 (7). This source seemed particularly promising because the cells had re- Abbreviations: GPI, glucosephosphate isomerase; IDH, isocitrate dehydrogenase. * Address reprint requests to this author. tained pluripotency, as seen in the tissues formed in solid growths, and because chromosomal examination disclosed that they were still very largely euploid (5, 6), as in the tumor of origin (8). In our first series of experiments (5, 6), small groups of five embryoid body core cells were injected into blastocysts. There they developed in a completely orderly fashion for the first time in their 8-year transplant history, and contrib- uted substantially to production of tumor-free healthy mice with a wide array of tumor-derived tissues, including some never seen in the solid tumors themselves. Adult tissue-spe- cific products (e.g., immunoglobulins, hemoglobin, liver proteins, melanin, etc.), of the genetic variant types charac- teristic of the inbred strain in which the tumor had origi- nated, but not of the blastocyst strain, attested to normal function. Cells derived from the teratoma, which in this case is of X/Y sex chromosome type, also gave rise to sperms from which many normal progeny were obtained. These re- sults strongly indicated that individual embryonal carcino- ma cells probably had the full range of developmental potentialities; and that teratocarcinogenesis entails changes in gene function rather than gene structure. In the present series of experiments, a final test of tera- toma-stem-cell totipotency was sought by introduction of single embryoid body core cells into blastocysts, to allow clo- nal propagation of the donor cell. Single-cell injections have the further advantage that they test the developmental con- sequences of any chromosomal or genetic variations among the source cells. We report here definitive evidence for de- velopmental totipotency of single teratocarcinoma cells. Pro- duction of teratoma-free animals with many tumor-derived normal tissues indicates that retention of euploidy in the tumor cells is a sufficient, and possibly a necessary, condi- tion for restoration of completely normal gene expression in an appropriate environment. MATERIALS AND METHODS Teratocarcinoma Cells. The OTT 6050 teratoma was ex- perimentally produced by Stevens in 1967 (7), by grafting a 6-day chromosomally male (X/Y) embryo (8) of the 129/Sv slJ C P inbred strain (to be referred to as 129) under the tes- tis capsule. The embryo became disorganized and formed a teratoma which, after intraperitoneal injection into another recipient, was converted to a modified ascites form. "Em- bryoid bodies" in the ascites consist, when small-size, of a "core" of embryonal carcinoma cells surrounded by a "rind" of yolk sac epithelium. The core cells were used for blasto- cyst injections. They were isolated, as before (6), from em- bryoid bodies of less than 100 ,um size, or from a modified source (to be described elsewhere) which yielded similar re- sults. Injections into Blastocysts. The microinjection proce- 549 Downloaded by guest on May 14, 2020
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Proc. Nat. Acad. Sci. USAVol. 73, No. 2, pp. 549-553, February 1976Cell Biology
Totipotency and normal differentiation of single teratocarcinomacells cloned by injection into blastocysts
(mouse teratoma/embryonal carcinoma/embryoid body core cells/allophenic mice/genetic mosaicism)
KARL ILLMENSEE AND BEATRICE MINTZ*Institute for Cancer Research, Fox Chase, Philadelphia, Pennsylvania 19111
Contributed by Beatrice Mintz, November 19,1975
ABSTRACT A definitive test for developmental totipo-tency of mouse malignant teratocarcinoma cells was con-ducted by cloning singly injected cells in genetically markedblastocysts. Totipotency was conclusively shown in an adultmosaic female whose tumor-strain cells had made substantialcontributions to all of the wide range of its somatic tissuesanalyzed; the clonally propagated cell lineage had thereforedifferentiated in numerous normal directions. The test cellswere from "cores" of embryoid bodies of a euploid, chromo-somally male (X/Y), ascites tumor grown only in vivo bytransplantation for 8 years. The capacity of cells from thesame source to differentiate, in a phenotypic male, into rep-roductively functional sperms, has been shown in our previ-ous experiments [(1975) Proc. Nat. Acad. Sci. USA 72, 3585-3589]. Cells from this transplant line therefore provide mate-rial suitable for projected somatic and germ-line geneticanalyses of mammalian differentiation based on "cycling" ofmutation-carrying tumor cells through developing embryos.In some animals obtained from single-cell injections, tumor-derived cells were sporadically distributed in developmental-ly unrelated tissues. These cases can be accounted for by de-layed and haphazard cellular integration, and by a markeddegree of sustained cellular developmental flexibility inearly mammalian development, irrespective of certain classi-cal "germ-layer" designations. All mosaic mice obtainedhave thus far been free of teratomas. In one case, the inject-ed stem cell contributed only to the pancreas and gave rise toa malignancy resembling pancreatic adenocarcinoma. Thehigh modal frequency of euploidy in these individually test-ed cells thus tends to indicate that a near-normal chromo-some complement is sufficient for total restoration of orderlygene expression in a normal embryonic environment; it mayalso be necessary for teratoma stem-cell proliferation to beterminated there.
Teratomas, unlike other tumors, often comprise a variety oftissues, derived from teratocarcinoma, or embryonal carci-noma, stem cells (1, 2). Pluripotency of the stem cells wasdemonstrated when solid tumors of varied tissue composi-tion were obtained from transfers of single cells to grafthosts (3). However, the consistent absence of certain tissues(4), and the immaturity and aberrations of others, in terato-mas has until recently left open the question whether em-bryonal carcinoma cells are in fact developmentally totipo-tent. Full realization of normal developmental potentialitieswould be expected to occur, for embryonal carcinoma as fornormal embryonal cells, only in the normally organized en-vironment of an early embryo. We therefore undertook ex-periments in which mouse teratocarcinoma cells were inject-ed into the cavity of genetically marked blastocysts (5, 6).The donor cells were from the "cores" of teratoma embryoidbodies grown only in vvo as a transplantable ascites sincethe tumor (OTT 6050) was isolated in 1967 (7). This sourceseemed particularly promising because the cells had re-
Abbreviations: GPI, glucosephosphate isomerase; IDH, isocitratedehydrogenase.* Address reprint requests to this author.
tained pluripotency, as seen in the tissues formed in solidgrowths, and because chromosomal examination disclosedthat they were still very largely euploid (5, 6), as in thetumor of origin (8).
In our first series of experiments (5, 6), small groups offive embryoid body core cells were injected into blastocysts.There they developed in a completely orderly fashion forthe first time in their 8-year transplant history, and contrib-uted substantially to production of tumor-free healthy micewith a wide array of tumor-derived tissues, including somenever seen in the solid tumors themselves. Adult tissue-spe-cific products (e.g., immunoglobulins, hemoglobin, liverproteins, melanin, etc.), of the genetic variant types charac-teristic of the inbred strain in which the tumor had origi-nated, but not of the blastocyst strain, attested to normalfunction. Cells derived from the teratoma, which in this caseis of X/Y sex chromosome type, also gave rise to spermsfrom which many normal progeny were obtained. These re-sults strongly indicated that individual embryonal carcino-ma cells probably had the full range of developmentalpotentialities; and that teratocarcinogenesis entails changesin gene function rather than gene structure.
In the present series of experiments, a final test of tera-toma-stem-cell totipotency was sought by introduction ofsingle embryoid body core cells into blastocysts, to allow clo-nal propagation of the donor cell. Single-cell injections havethe further advantage that they test the developmental con-sequences of any chromosomal or genetic variations amongthe source cells. We report here definitive evidence for de-velopmental totipotency of single teratocarcinoma cells. Pro-duction of teratoma-free animals with many tumor-derivednormal tissues indicates that retention of euploidy in thetumor cells is a sufficient, and possibly a necessary, condi-tion for restoration of completely normal gene expression inan appropriate environment.
MATERIALS AND METHODSTeratocarcinoma Cells. The OTT 6050 teratoma was ex-
perimentally produced by Stevens in 1967 (7), by grafting a6-day chromosomally male (X/Y) embryo (8) of the 129/SvslJ C P inbred strain (to be referred to as 129) under the tes-tis capsule. The embryo became disorganized and formed ateratoma which, after intraperitoneal injection into anotherrecipient, was converted to a modified ascites form. "Em-bryoid bodies" in the ascites consist, when small-size, of a"core" of embryonal carcinoma cells surrounded by a "rind"of yolk sac epithelium. The core cells were used for blasto-cyst injections. They were isolated, as before (6), from em-bryoid bodies of less than 100 ,um size, or from a modifiedsource (to be described elsewhere) which yielded similar re-sults.
Injections into Blastocysts. The microinjection proce-
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Table 1. Tissue contributionsa clonally derived from single teratocarcinoma cells (129 strain)after injection into blastocysts (WH or C57BL/6-b/b strain)
a Coat strain-specific markers were at the agouti locus (in injections into WH blastocysts), expressed in hair follicle dermis, or at both theagouti locus and the black locus, expressed in melanocytes (in C57BL/6-b/b blastocyst injections). All other tested tissues were biochemi-cally analyzed for GPI electrophoretic allelic strain variants. In addition, some tests (italics, second line of case B) were conducted for IDHaftelic variants, because absence of IDH expression in blood cells permits blood contamination to be ruled out as a source of any 129-straincomponent in-IDH-tested tissues. All data are given in percent 129 type.Salivary glands, 40.
c Salivary glands, 5.d (Hepatitis).e Placenta, 0.f (W/W anemia) .
NCarcass, 0 (W/W anemia) .
h Pancreatic adenocarcinoma, 100. The pancreatic tumor found in this animal had some histologically normal elements, and the homogenatecontained 50% of each GPI strain (Fig. 5, slot b). The tumor grew onl/ in 129-strain transplant hosts. Therefore,sthe actual cancerous com-ponent was apparently all of the histocompatible 129 strain-type (i.e., teratocarcinoma-derived) as shown in the corrected entry (100%) inthe table, and the normal component was blastocyst-derived. A small amount of ostensibly normal pancreas was included with stomach andintestines in the GPI test of a homogenate of those tissues; the 5% 129-strain GPI in that test was probably due only to some accompanyingpancreatic adenocarcinoma.
dure (9) was again used to entrap a teratocarcinoma cell inthe blastocyst cavity near the inner cell mass. Most recipientblastocysts were of the WH (ICR subline) inbred strain, inwhich only the W (dominant white spotting) locus is segre-gating; matings of +/W parents were used. Both +/+ and+/W segregants are normal; W/W individuals ordinarilydie of a severe anemia within a few days after birth. A fewblastocysts were of the C57BL/6-b/b strain (to be referredto as C57-b/b), which differs from C57BL/6 only by muta-tion of the black (B) coat-color allele to brown (b). Injectedblastocysts, after a few hours' incubation (6), were surgicallytransferred to uteri of pseudopregnant ICR albino females.
Tissue Genotypic Analyses. The 129 strain of tumor ori-gin is black (B/B) for a melanocyte marker and white-bel-lied agouti (AW/Aw) for a dermal hair-follicle marker. TheWH blastocyst strain is black and non-agouti (a/a); theC57-b/b blastocyst strain is brown (b/b) and non-agouti.With the glucosephosphate isomerase (GPI; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) marker, tumor- andblastocyst-derived cells can be distinguished in blood celllysates and in homogenates of soft tissues, by starch gel elec-trophoresis (10) of allelic forms (Gpi-1 locus). The 129 strain
is homozygous for the slow-migrating Gpi-la type, WH andC57-b/b strains for the fast Gpi-lb type. Another marker,NADP-dependent isocitrate dehydrogenase [IDH; threo-Ds-isocitrate:NADP+ oxidoreductase (decarboxylating), EC1.1.1.42], which is not expressed in blood cells, was also usedfor independent tests of some tissue homogenates, by starchgel electrophoresis (11). The 129 strain is homozygous forthe slow-migrating Id-la variant, the WH and C57-b/bstrains for the fast-migrating Id-lb type.
RESULTSOf a total of 161 injected and surgically transferred blasto-cysts, 71 (44%) survived. Three were from C57-b/b- and 68from WH-strain blastocysts. The survivors (33 females, 34males, four of unrecorded sex) included five living fetusessacrificed on day 15 of gestation; 16 live animals deliveredby caesarean section at term, for genotypic comparisonswith their placentas, and sacrificed within a day's time; and50 older postnatal animals. Of the 50 postnatal animals, 24were autopsied at 1-7 weeks of age; the remaining 26 arestill alive and now range from 9 to 16 weeks of age. Analyses
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.4~~~.1 0W*4t 04
Table 2. Placenta compared with total-bodycontributions* (estimated from percent 129-strain-type
GPI) clonally derived from single 129-strain teratocarcinomacells after injection into WH-strain blastocysts
a b c d e f g h i j k IFIG. 1. GPI allelic strain variants in starch gel electrophoresis
of tissue homogenates. Slot (a), a 1:1 mixture of controls: 129-strain-type, slow-migrating; WH type, fast-migrating. Tissuesfrom experimental animal A: blood (b), brain (c), spleen (d), heart(e), skeletal muscle, showing a hybrid enzyme band due to hetero-karyon formation (f), kidneys (g), reproductive tract (h), liver (i),pooled stomach, intestines, and pancreas (j), thymus (k), and lungs(1). The 129-strain component of all these normal tissues was de-rived from a single teratocarcinoma cell after injection into a WH-strain blastocyst. (Origin and anode are lowermost.)
have been completed on all except the 26 living animals,which have had only coat and blood cells genotypically clas-sified; five have coat and/or blood mosaicism and some ofthe remaining 21 may prove later to have some internal tis-sue mosaicism. Among all 71 animals produced, whetherpartially or completely analyzed, there were 21 (30%) with129-strain cells in one or more tissues (Tables 1 and 2). Inaddition, six resorption sites were found during caesarean
sections; with the GPI marker, they had 0, 40%, 75%, 80%,80%, and 85%, respectively, of the 129 type. Histologicalsamples were indistinguishable from those in spontaneousresorptions among controls.The results summarized in Table 1 clearly demonstrate
that the clonal lineage of a single embryonal carcinoma cellfrom an embryoid body core can differentiate in widely di-verse directions, contributing to all tissues thus far tested, as
in case A (Fig. 1). Inasmuch as GPI is expressed in bloodcells as well as other tissues which themselves contain blood,it should be emphasized that this marker alone does notprove mosaicism in tissues other than blood, if the animalalso has blood mosaicism. Evidence for tissue-specific mosai-cism was therefore obtained from the following sources: (i)the 129 type of GPI was absent from the bloods of severalanimals but present, unequivocally, in their other tissues(cases C, D, E, H, I, and N). (ii) Quantitative estimates ofGPI isozymes indicated that a number of tissues possessedthe 129-strain GPI type at appreciably higher levels than ac-
countable for by that animal's blood (e.g., 50% 129 type inthe lungs, and only 10% in the circulating blood, of animalB) (Fig. 2). (iii) IDH, which is not expressed in blood cells,was used as an independent marker in tissue homogenatesfrom cases A and B (Table 1 and Fig. 3), where it confirmedthe presence of tissue-specific 129-strain contributions.(IDH-isozyme estimates for case A are omitted from Table 1
Case Sex Placentat Body
P 6 50 l0tQ d 40 40R i 40 25S 9 10 75T d 10 33
*' From cases delivered by caesarean section at term (P-S) or on day15 of gestation (T). An additional placenta (U) had 50% 129-typeGPI; the animal itself was cannibalized.
t Each surrogate mother had only the WH-strain GPI type and,therefore, did not contribute any of the 129-strain placental cells.The bloods of animals Q and S had only WH-type GPI and alsodid not account for any of their placental 129-type GPI.
t W/Wanemia was present in this animal.
because the gels were technically inadequate for quantita-tion.) (iv) In skeletal muscle, the occurrence of an interme-diate or hybrid enzyme band in both the GPI and IDH testsproved that cells of both strains had specifically differentiat-ed as myoblasts and had fused and formed heterokaryonswith heterodimeric enzyme, as previously shown in muscleof allophenic mice (11). (v) Coat mosaicism in four animals(all from WH-strain blastocysts) provided further evidencefor a tissue-specific, as well as a normally functioning, 129-strain contribution, by means of allelic differences at the ag-outi locus, expressed in hair follicles. The 129-strain elementin the coats was notably small, comprising only a few abbre-viated agouti stripes or patches on the face (cases A and G)or dorsum (cases M and L) on a non-agouti ground. (vi) Dif-ferentiation into placental tissues also occurred (Table 2).The 129-type GPI placental contribution was shown in allcases (P-U) not to be of maternal origin (Fig. 4) and, in atleast two cases (Q and S), not to be due to blood from thefetus.No animal has thus far shown any indication of a terato-
ma. This confirms the results of our previous study in which93 survivors, from blastocysts injected with groups of fiveembryoid body core cells, were also free of teratomas (ref. 6,and unpublished results). Among the few ailments was acase (animal C) of severe diffuse hepatitis resembling thenot uncommon disease caused by mouse hepatitis virus.There is no reason to ascribe the condition to the 10% of theliver derived from the teratoma cell. There were five casesof genetically caused W/W anemia (from injected blasto-cysts of +/W parents). Three of these animals (Tables 1 and
a b c d e f g h i j k I m a b c d e f g h iFIG. 2 (left). GPI strain-specific allelic variants in starch gel electrophoresis of tissue homogenates. Slot (a), a 1:1 mixture of 129 (slow-
migrating-type) and WH (fast-iype) controls. Tissues from normal experimental animal B: blood (b), brain (c), spleen (d), heart (e), skeletalmuscle (f), kidneys (g), reproductive tract (h), liver (i), pooled stomach, intestines, and pancreas (j), thymus (k), lungs (1), and salivary glands(m). All 129-type contributions arose from one teratocarcinoma cell placed in a WH blastocyst.
FIG. 3 (right). Verification of specific-tissue mosaicism, irrespective of blood content, of the same animal as in Fig. 2, by means of an in-dependent marker not expressed in blood. IDH allelic strain variants in starch gel electrophoresis of tissue homogenates. Slot (a), a 1:1 mix-ture of 129 (slow-migrating-type) and WH (fast-type) controls. The experimental animal's tissues are brain (b), spleen (c), heart (d), kidneys(e), liver (f), thymu's (g), lungs (h), and salivary glands (i).
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a b c d e f 9 h i i
FIG. 4. Comparisons of placenta and whole-body strain com-
position (including blood cells in each) after injection of single ter-atocarcinoma cells (129 strain) into blastocysts (WH strain) fol-lowed by caesarean delivery at term. GPI isozymic strain variantsin starch gel electrophoresis of tissue homogenates from 129-strain(slot a) and WH-strain (b) controls and a 1:1 control mixture (c),and from the placenta (d) and body (e) of case S; placenta (f) andbody (g) of case Q; and placenta (h) and body (i) of case P. Thesurrogate mother of the first case (j) and each of the other cases
was of the fast GPI type and therefore did not contribute to the129-strain GPI in the placenta.
2) proved to have 129-type cells which, in at least one case
(F), were identified in the blood. (A fourth W/W had onlyblastocyst-derived cells, and the fifth was cannibalized.)The sole instance of an abnormality directly attributable
to cells from the teratoma lineage was striking and withoutprecedent. Animal 0, obviously ill and retarded since birth,was killed at 2 weeks of age and found to have a conspicuouspancreatic tumor. In histological examination, kindly per-
formed by Dr. R. P. Custer of this Institute, it resembled a
pancreatic adenocarcinoma, predominantly of the acinartype but also with ductal epithelial-dysplasia resembling car-
cinoma in situ. During the autopsy, pieces were grafted sub-cutaneously into recipients of the C57BL/6-b/b strain, fromwhich the blastocyst had been obtained, and the 129 tera-toma-strain; these have some histocompatibility allelic dif-ferences at the major locus (H-2b and H-2bc, respectively)(12), and at minor loci. Three of four grafts grew in 129-strain hosts; none of five grafts survived in C57-b/b hosts.Therefore, although a homogenate of the tumor contained50% of each GPI strain-type (Fig. 5, slot b), the actual malig-nant component was apparently all of the 129 strain (Table1). This conclusion is consistent with the fact that the tumormass included some histologically normal elements, proba-bly largely of C57-b/b strain composition, as judged fromthe GPI results. The animal had no other tumors and hadonly C57-b/b cells in its other tissues. (A very slight amountof 129-type GPI in pooled stomach, intestines, and an osten-
sibly normal part of the pancreas was, in all likelihood, dueto contamination with some 129-strain pancreatic tumor
cells.)
DISCUSSIONA conclusive demonstration of teratocarcinoma-cell develop-mental totipotency would require that a single cell be shownto form all fully differentiated somatic tissues. The singleembryoid body core cell injected into a blastocyst in case A(Table 1 and Fig. 1) has now provided such evidence, basedon examination of virtually all major tissues in the resultantanimal. The data thus support and extend our earlier conclu-sion of probable totipotency, from experiments in whichgroups of five embryoid body core cells were introducedinto blastocysts (5, 6). Cells derived from this X/Y tumor can
also form fully functional sperms in a phenotypic male (6).From evidence in X/X 4-* X/Y allophenic mice (13), X/Y
primordial germ cells would not be expected to progress to
the gamete stage in a phenotypic female such as animal A.In the strict sense, a functional germ-line contribution maynot be essential for proof of totipotency: totipotent embryo
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a b c d e f g h
FIG. 5. GPI strain-specific variants in starch gel electrophore-sis of tissue homogenates from a 1:1 mixture of 129 (slow-migrat-ing-type) and C57BL/6-b/b (fast-migrating) controls (slot a) andof a tumor (b) found in the pancreas of animal 0, obtained afterinjection of one 129-strain teratocarcinoma cell into a C57BL/6-b/b blastocyst. Histologically, the tumor mass contained both ma-lignant and normal cells; transplant tests showed the malignantcomponent to be all of the 129 strain. All other tissues tested weresolely blastocyst-derived; they included blood (c), brain (d), skele-tal muscle (e), kidneys (f), liver (g), and lungs (h).
cells of certain sterile genotypes (e.g., W/W) form micewith all somatic tissues but a deficiency of germ cells. Nev-ertheless, possession by teratoma cells of the capacities forfull germ-line (6) as well as somatic differentiation is fortu-nate, as both are indispensable for realizing one of the long-range aims underlying this work.The aim alluded to, and outlined earlier (5, 6), is to utilize
mutation-carrying teratocarcinoma cells as a new experi-mental tool for the analysis of mammalian differentiation invivo, by "cycling" them through mice via blastocyst injec-tions. Presumptive or known mutations could then be as-sessed for full developmental and biochemical expression inthe soma; the heritable status of the variant could be shownby its transmission to progeny; and its location in relation toother genes could be mapped through recombination in thegerm-line.
Inclusion of some blastocysts of the lethally anemic W/Wgenotype in the present work was intended as an explorationof one facet of these projected genetic studies: the use of le-thal genes. Most lethals are probably deleterious becauseonly one or a few, rather than all, cell types are defective.Replacement or interaction of the defective cells with genet-ically normal ones could permit the animal to be "rescued"and the analyses to go forward. An example of rescue byspecific-cell replacement is the fully viable W/W , + / +allophenic mouse, in which sufficient normal (+1+) embryocells have occupied the hematopoietic system (14). The sameprinciple is being tested here, albeit in some future experi-ments the lethal genes may be introduced in the teratomacells. Three mice from injected W/W blastocysts were infact found to have tumor-derived cells (Tables i and 2),identified in at least one case (F) as having made some con-tribution to the blood. Since the animal was killed for study,its chances for survival are unknown, but the results are en-couraging for the further use of lethal-normal combinations.
In both our 5-cell and our 1-cell injection series, the initialnumber of teratoma cells is close to the 3-cell number esti-mated (14) to comprise the precursor cells of the embryoproper, as distinct from future extraembryonic components.Yet the 5-cell injections yielded 16% (15/93) of the survivorswith teratoma-derived cells (ref. 6, and unpublished data),the 1-cell injections at least 30% (21/71, with 21 living micealmost wholly unanalyzed). While some of the increase maybe attributable to improved technique (reflected in the 33%compared to 44% survival rates), it is probably due chiefly tothe observed relatively greater tendency for a single injectedcell to remain near the inner cell mass when deposited there,hence to be integrated into the embryo-forming region.
Cases of sporadic distribution of clonally derived 129-strain cells, often in developmentally unrelated tissues
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(Table 1), are strikingly more frequent than previously ob-served in conventional allophenic mice from aggregates oftwo blastomere strains (14 16). This disparity could conceiv-ably result from a tendency toward delayed teratoma-cellintegration into the embryo, perhaps due to initial differ-ences in donor and host cellular adhesiveness (5). There mayby then be a small donor clone whose cells are scattered torelatively few places, where development proceeds accord-ing to local cues. This could also account for the low inci-dence and small-area coverage of coat mosaicism (Table 1),again in contrast to allophenic mice. Melanocyte determina-tion in the mouse [from 17 pairs of neural crest cells (17)]and hair-follicle-dermis determination [from some 85 pairsof somite cells (14)] are believed to occur on about late day 6of embryonic life, in precursor cells flanking the mid-dor-sum and strung out along the full length of the embryo. If infact a teratoma cell injected into a day 3 blastocyst tendsoften to undergo late assimilation and limited distribution,its daughter cells would be least often represented in thosetissues, such as the coat, that originate from a relatively far-flung system of specific precursor cells. Coat markers maytherefore be the least favorable ones to reveal teratoma-cellparticipation. This is consistent with the results of Brinster(18), who, in the only previous attempt to inject embryonalcarcinoma cells into blastocysts, used coat color as the onlymarker (thereby precluding tests for totipotency). He ob-tained only one animal, out of 60 survivors, with a slighttumor-strain contribution in its coat.The frequent sporadic distribution of tumor-derived
tissues (Table 1) also serves as a caveat for future experi-ments with mutagenized teratocarcinoma cells: in order todemonstrate any specific "restriction" in differentiation ofthese cells, that could be attributed to their mutant status, itwould first be necessary to survey a large sample of mosaicanimals.
Prolonged capacity of tumor-derived cells to undergo assi-milation and subsequent differentiation also implies that thedonor cells had continued to remain totipotent for sometime. This would not be surprising, in view of the experi-mental origin of the teratoma from a 6-day postimplantationembryo (7). Moreover, the tumor stem cells have been foundto resemble certain undifferentiated cells of the early post-implantation stage, rather than preimplantation-stage cells,in their ultrastructure (19) and alkaline phosphatase content(20); their soluble protein profiles also differ from those ofmorula cells (5). The evidence thus strongly supports theprobability that some totipotent cells are normally stillpresent as late as day 6 of embryonic life. The candidate to-tipotent cells have been tentatively identified as "ecto-meso-derm" (19, 20), according to conventional "germ-layer" ter-minology. While germ layers normally arise in characteristiclocations (for still unknown reasons) and normally undergoorderly movements ending in specific differentiations, ex-periments with many species (21), including mammalianembryos (22), have long shown that layers are substantiallyinterconvertible and capable of giving rise to structures usu-ally formed from the others. Totipotency of individual"ecto-mesoderm" cells is consistent with such flexibility.The only abnormal derivative obtained here was the pan-
creatic tumor in case 0. Localization of tumor-strain cellsand tumor specifically to the pancreas may possibly signifythat the single donor cell was already restricted to the status
of a pancreatic stem cell which either had an intrinsic de-fect, or else underwent de novo neoplastic conversion. Thiscase demonstrates that the requirement that single cells beinjected into blastocysts, for proof of developmental totipo-tency, is realistic, as some specialized types of stem cells orsome irreversibly malignant cells may be included if largenumbers of cells are introduced.
Note Added in Proof. In a recent report from another laboratory[Papaioannou, V., McBurney, M., Gardner, R. & Evans, M. (1975)Nature 258, 70-73], blastocysts were injected with 20 to 40 terato-carcinoma cells each, from three in vitro cell lines from other tera-tomas. Of a total of 11 mosaic mice with several somatic tissue con-tributions from the injected cells, as judged from GPI and pigmentmarkers, most of the animals, obtained from two of the cell lines,also developed tumors. The remaining line, characterized by 40chromosomes and X/O sex chromosome constitution, yielded onemouse without a tumor.
This work was supported by USPHS Grants HD-01646, CA-06927, and RR-05539, and by an appropriation from the Common-wealth of Pennsylvania. We thank Mrs. Claire Cronmiller for excel-lent technical assistance.
1. Stevens, L. C. (1967) Adv. Morphog. 6, 1-31.2. Pierce, G. B. (1967) in Current Topics in Developmental
Biology, eds. Moscona, A. A. & Monroy, A. (Academic Press,New York), Vol. 2, pp. 223-246.
3. Kleinsmith, L. J. & Pierce, G. B., Jr. (1964) Cancer Res. 24,1544-1551.
4. Stevens, L. C. & Hummel, K. P. (1957) J. Nat. Cancer Inst.18,719-747.
5. Mintz, B., Illmensee, K. & Gearhart, J. D. (1975) in Symp. onTeratomas and Differentiation, eds. Sherman, M. I. & Solter,D. (Academic Press, New York), 59-82.
6. Mintz, B. & Illmensee, K. (1975) Proc. Nat. Acad. Sci. USA72,3585-3589.
7. Stevens, L. C. (1970) Dev. Biol. 21, 264-382.8. Dunn, G. R. & Stevens, L. C. (1970) J. Nat. Cancer Inst. 44,
99-105.9. Lin, T. P. (1966) Science 151,333-337.
10. Gearhart, J. D. & Mintz, B. (1972) Dev. Biol. 29, 27-37.11. Mintz, B. & Baker, W. W. (1967) Proc. Nat. Acad. Sci. USA
58,592-598.12. Snell, G. D., Graff, R. J. & Cherry, M. (1971) Transplanta-
tion. 11,525-530.13. Mintz, B. (1969) in Birth Defects: Original Article Series 5
(National Foundation, New York), pp. 11-22.14. Mintz, B. (1970) in Symp. Int. Soc. Cell Biol., ed. Padykula,
H. (Academic Press, New York), Vol. 9, pp. 15-42.15. Mintz, B. (1971) in Symp. Soc. Exp. Biol., eds. Davies, D. D. &
Balls, M. (Cambridge University Press, New York), Vol. 25,pp. 345-370.
16. Mintz, B. (1974) Annu. Rev. Genet. 8,411-470.17. Mintz, B. (1967) Proc. Nat. Acad. Sci. USA 58,344-351.18. Brinster, R. L. (1974) J. Exp. Med. 140, 1049-1056.19. Damjanov, I., Solter, D., Belicza, M. & Skreb, N. (1971) J.
Nat. Cancer Inst. 46,471-480.20. Damjanov, I., Solter, D. & Skreb, N. (1971) Z. Krebsforsch. 76,
249-256.21. Oppenheimer, J. M. (1940) Quart. Rev. Biol. 15, 1-17.22. Levak-Svajger, B. & Svajger, A. (1974) J. Embryol. Exp. Mor-
