Interactions between diploid embryonal carcinoma cells and early embryonic cells

Two diploid embryonal carcinoma (EC) cell lines, P10 and P19, differ in their response to the embryonic environment. P10 produces mostly normal chimeras following injection into blastocysts, whereas P19 produces mostly abnormal chimeras. In this study, P10 cells were aggregated with morulae, and all resulting fetuses were chimeric with very large contributions from the EC cells. However, all embryos were abnormal. Following aggregation of P19 cells with morulae, very few embryos were recovered and they were all non-chimeric. Both P10 and P19 were capable of forming functional gap junctions with morula cells and with the ICM of the blastocyst but not with trophoblast, showing that differences in the ability to make junctional contact with the embryo cannot explain the differences between the two cell lines.     

Generating Porcine Chimeras Using Inner Cell Mass Cells and Parthenogenetic Preimplantation Embryos

Background

The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells.

Methodology/Significant Principal Findings

In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4–8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4–8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively.

Conclusion/Significance

Our findings indicate that the aggregation method using parthenogenetic morulae or 4–8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras.     

Embryonic stem cells alone are able to support fetal development in the mouse

The developmental potential of embryonic stem (ES) cells versus 3.5 day inner cell mass (ICM) was compared after aggregation with normal diploid embryos and with developmentally compromised tetraploid embryos. ES cells were capable of colonizing somatic tissues in diploid aggregation chimeras but less efficiently than ICMs of the same genotype. When ICM in equilibrium with tetraploid and ES in equilibrium with tetraploid chimeras were made, the newborns were almost all completely ICM- or ES-derived, as judged by GPI isozyme analysis, but tetraploid cells were found in the yolk sac endoderm and trophectoderm lineage. Investigation of ES contribution in 13.5 day ES in equilibrium with tetraploid chimeras by DNA in situ hybridization confirmed the complete tetraploid origin of the placenta (except the fetal blood and blood vessels) and the yolk sac endoderm. However, the yolk sac mesoderm, amnion and fetus contained only ES-derived cells. ES-derived newborns failed to survive after birth, although they had normal birthweight and anatomically they appeared normal. This phenomenon remains unexplained at the moment. The present results prove that ES cells are able to support complete fetal development, resulting in ES-derived newborns, and suggest a useful route for studying the development of genetically manipulated ES cells in all fetal lineages.     

Pluripotency of cultured rabbit inner cell mass cells detected by isozyme analysis and eye pigmentation of fetuses following injection into blastocysts or morulae

Pluripotency of isolated rabbit inner cell masses (ICMs) and cultured (3 days) inner cell mass (ICM) cells was tested by injecting these donor cells into day 3.5 blastocysts (experiment 1) or day 3 morulae (experiment 2) to produce chimeric embryos. Injected (n = 107) and noninjected (n = 103) embryos were transferred to the opposite uterine horns of the same recipient females. Chimerism was determined by adenosine deaminase (ADA) isozyme analysis on fetal tissue and by eye pigmentation at midgestation. In experiment 1, 53% and 64%, respectively, of blastocysts injected with ICMs or cultured ICM cells developed to midgestation, compared with 52% and 48% for controls. Of these fetuses, four (31%) and one (6%), respectively, had ADA chimerism. In experiment 2,38% and 62%, respectively, of the morulae injected with ICMs or cultured ICM cells developed to midgestation, compared with 46% and 56% for control morulae. Six (43%) chimeric fetuses from morulae injected with ICMs were detected by ADA analysis, but 12 (86%) chimeric fetuses were detected by eye pigmentation, indicating that eye pigmentation was a more sensitive marker for chimerism than our ADA assay. None of the 14 fetuses recovered after injecting morulae with cultured ICM cells were chimeric with either marker. No chimeras developed from control embryos. These studies demonstrate (1) that pregnancy rates are not compromised by injection of blastocysts or morulae with ICMs or cultured ICM cells, (2) that chimeric rabbit fetuses can be produced by injecting ICMs into either blastocysts or morulae, and (3) that cultured ICM cells can contribute to embryonic development when injected into blastocysts. © 1993 Wiley-Liss, Inc.     

In vitro formation of tetraploid rat blastocysts after fusion of two-cell embryos

Gene targeting technology is not available in the rat which is an animal model of major importance, e.g., in cardiovascular research. This is due to the fact that the rat embryonic stem cell (ESC)-like cells established by several groups do not form germ-line chimeras when injected into blastocysts. In the mouse, the aggregation of ESC with tetraploid embryos has allowed the generation of animals completely derived from these cells. However, aggregation of rat ESC-like cells with tetraploid rat embryos has not yet been attempted to evaluate their developmental capacity. Therefore, we established a method to produce tetraploid rat embryos by fusion at the two-cell stage. Chemical fusion by polyethylene glycol (PEG) was shown to be less efficient (56.3% fused embryos) than electrofusion (96.1% fused embryos). The rate of development of fused embryos to blastocysts was independent of the fusion method and similar to the rate of control embryos. However, this rate was lower when the embryos had been cultured from the zygote state before fusion (14-20%) compared to freshly isolated two-cell embryos (41-63%). Alike for the mouse, blastocysts derived from fused two-cell rat embryos contained about half the number of cells as control blastocysts and were homogeneously tetraploid with no evidence of mosaicism. This method may be useful for the establishment of gene-targeting technology in the rat.     

Embryonic stem cells contribute to mouse chimeras in the absence of detectable cell fusion

Embryonic stem (ES) cells are capable of differentiating into all embryonic and adult cell types following mouse chimera production. Although injection of diploid ES cells into tetraploid blastocysts suggests that tetraploid cells have a selective disadvantage in the developing embryo, tetraploid hybrid cells, formed by cell fusion between ES cells and somatic cells, have been reported to contribute to mouse chimeras. In addition, other examples of apparent stem cell plasticity have recently been shown to be the result of cell fusion. Here we investigate whether ES cells contribute to mouse chimeras through a cell fusion mechanism. Fluorescence in situ hybridization (FISH) analysis for X and Y chromosomes was performed on dissociated tissues from embryonic, neonatal, and adult wild-type, and chimeric mice to follow the ploidy distributions of cells from various tissues. FISH analysis showed that the ploidy distributions in dissociated tissues, notably the tetraploid cell number, did not differ between chimeric and wild-type tissues. To address the possibility that early cell fusion events are hidden by subsequent reductive divisions or other changes in cell ploidy, we injected Z/EG (lacZ/EGFP) ES cells into ACTB-cre blastocysts. Recombination can only occur as the result of cell fusion, and the recombined allele should persist through any subsequent changes in cell ploidy. We did not detect evidence of fusion in embryonic chimeras either by direct fluorescence microscopy for GFP or by PCR amplification of the recombined Z/EG locus on genomic DNA from ACTB-cre::Z/EG chimeric embryos. Our results argue strongly against cell fusion as a mechanism by which ES cells contribute to chimeras.     

In vitro development and cell allocation following aggregation of split embryos with tetraploid or developmentally asynchronous blastomeres in rhesus monkeys

Production of genetically identical pairs of monkeys would have tremendous implications for biomedical research, particularly immunological studies and vaccine trials. Specific aims of this study were to (1) determine whether aggregation of embryos split into halves or quarters with equal numbers of either developmentally asynchronous or tetraploid blastomeres would enhance their developmental potential in vitro and increase total cell numbers in resulting blastocysts, and (2) determine the allocation of tetraploid and developmentally asynchronous blastomeres in resulting blastocysts. Results demonstrated that development into blastocysts was greater (p < 0.05) for embryos split into pairs (39.8%) than for those split into quadruplet sets (17.4%) and similar (p > 0.05) to that of nonmanipulated controls (59.6%). Creation of chimeras from aggregation of a single 4-cell and four 16-cell stage blastomeres resulted in blastocyst formation (69.2%) similar to that of nonmanipulated control embryos (66.9%). However, neither development nor total cell numbers in resulting blastocysts differed between aggregate chimeras and those split into quadruplet sets at the 16-cell stage. Blastocysts resulting from the aggregate chimeras were derived strictly from the 16-cell stage blastomeres, with complete exclusion of the 4-cell stage blastomeres. Aggregation of split embryos with equal numbers of tetraploid blastomeres doubled (p < 0.05) both the proportion developing into blastocysts and the total cell numbers in resulting blastocysts. Tetraploid blastomeres were allocated to both the inner cell mass and trophectoderm of resulting blastocysts. In conclusion, due to exclusion of the less advanced cells, aggregation of developmentally asynchronous blastomeres did not improve the developmental competence or cell numbers of split rhesus embryos. Reconstitution of split embryos with equal numbers of tetraploid blastomeres enhanced their developmental potential and cell numbers in resulting blastocysts. However, tetraploid blastomeres were allocated to both the inner cell mass and trophectoderm.     

Completely ES Cell-Derived Mice Produced by Tetraploid Complementation Using Inner Cell Mass (ICM) Deficient Blastocysts

Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs) by injection of diploid (2n) ESCs into tetraploid (4n) blastocysts (ESC-derived mice). This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS) cells. However, the underlying mechanism(s) of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM). We designate these as type a (presence of ICM at blastocyst stage) or type b (absence of ICM). ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.     

Developmental potential and behavior of tetraploid cells in the mouse embryo

Tetraploid (4n) mouse embryos die at variable developmental stages. By examining 4n embryos from F2 hybrid and outbred mice, we show that 4n developmental potential is influenced by genetic background. The imprinted inactivation of an X chromosome-linked eGFP transgene in extraembryonic tissues occurred correctly in 4n embryos. A decrease of the cleavage rate in 4n preimplantation embryos compared to diploid (2n) embryos was revealed by real-time imaging, using a histone H2b:eGFP reporter. It has previously been known that mouse chimeras produced by the combination of diploid (2n) embryos with embryonic stem (ES) cells result in mixtures of the two components in epiblast-derived tissues. In contrast, the use of 4n host embryos with ES cells restricts 4n cells from the embryonic regions of chimeras, resulting in mice that are believed to be completely ES-derived. Using H2b:eGFP transgenic mice and ES cells, the behavior of 4n cells was determined at single cell resolution in 4n:2n injection and aggregation chimeras. We found a significant contribution of 4n cells to the embryonic ectoderm at gastrulation in every chimera analyzed. We show that the transition of the embryonic regions from a chimeric tissue to a predominantly 2n tissue occurs after gastrulation and that tetraploid cells may persist to midgestation. These findings suggest that the results of previously published tetraploid complementation assays may be influenced by the presence of tetraploid cells in the otherwise diploid embryonic regions.     

Attempts to enhance production of porcine chimeras from embryonic germ cells and preimplantation embryos

Porcine embryonic germ (EG) cells share common features with porcine embryonic stem (ES) cells, including morphology, alkaline phosphatase activity and capacity for in vitro differentiation. Porcine EG cells are also capable of in vivo development by producing chimeras after blastocyst injection; however, the proportion of injected embryos that yield a chimera and the proportion of cells contributed by the cultured cells in each chimera are too low for practical use in genetic manipulation. Moreover, somatic, but not germ-line chimerism, has been reported from blastocyst injection using porcine ES or EG cells. To test whether efficiency of chimera production from blastocyst injection can be improved upon by changing the host embryo, we used as host embryos four groups according to developmental stage or length in culture: fresh 4-cell and 8-cell stage embryos subsequently cultured into blastocysts, fresh morulae, fresh blastocysts, and cultured blastocysts. Injection and embryo transfer of fresh and cultured blastocysts produced similar percentages of live piglets (17% versus 19%). Four piglets were judged to have a small degree of pigmentation chimerism, but microsatellite analysis failed to confirm chimerism in these or other piglets. Polymerase chain reaction analysis for detection of the porcine SRY gene in female piglets born from embryos injected with male EG cells identified six chimeras, at least one, but not more than two, from each treatment. Chimerism was confirmed in two putative pigmentation chimeras and in four piglets without overt signs of chimerism. The low percentage of injected embryos that yielded a chimera and the small contribution by EG cells to development of each confirmed chimera indicated that procedural changes in how EG cells were combined with host embryos were unsuccessful in increasing the likelihood that porcine EG cells will participate in embryonic development. Alternatively, our results suggested that improvements are needed in EG cell isolation and culture procedures to ensure in vitro maintenance of EG cell developmental capacity.     

Comparison of tetraploid blastocyst microinjection of outbred Crl:CD1(ICR), hybrid B6D2F1/Tac, and inbred C57BL/6NTac embryos for generation of mice derived from embryonic stem cells

Embryo electrofusion and tetraploid blastocyst microinjection is a modification of the traditional embryonic stem cell (ES cell)-based method to generate targeted mutant mice. Viability of tetraploid embryos is reportedly lower than with diploid embryos, with considerable interstrain variation. Here we assessed fetus and pup viability after ES cell microinjection of tetraploid blastocysts derived from outbred, hybrid, and inbred mice. Two-cell mouse embryos (C57BL/6NTac [B6], n = 788; B6D2F1/Tac [BDF1], n = 1871; Crl:CD1(ICR) [CD1], n = 1308) were electrofused; most resultant tetraploid blastocysts were injected with ES cells and surgically transferred into pseudopregnant recipient mice. Reproductive tracts were examined at midgestation for embryologic studies using B6 and BDF1 blastocysts; implantation sites and viable fetuses were counted. Pregnancies were carried to term for studies of targeted mutant mice using BDF1 and CD1 blastocysts, and pup yield was evaluated. Electrofusion rates of 2-cell embryos did not differ among B6, BDF1, and CD1 mice (overall mean, 92.8% +/- 5.4%). For embryologic studies, 244 B6 blastocysts were surgically transferred and 1 fetus was viable (0.41%), compared with 644 BDF1 blastocysts surgically transferred and 88 viable fetuses (13.7%). For targeted mutant mouse studies, 259 BDF1 blastocysts were surgically transferred yielding 10 pups (3.9%); 569 CD1 blastocysts yielded 44 pups (7.7%).     

Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.     

Survival of porcine inner cell masses in culture and after injection into blastocysts

Isolation of embryonic stem cells has been documented only in the mouse and perhaps the hamster and cow. We report results of experiments designed to determine the effect of age of porcine embryos (6 through 10 d after the first day of estrus) on isolation of cell lines with embryonic stem cell-like morphology. The capacity of fresh and short-term cultured inner cell mass (ICM) cells to differentiate into normal tissues after injection into blastocysts was also measured. Few Day-6 ICM survived in culture to the first passage onto fresh feeder cells, but cell lines with embryonic stem cell-like morphology developed from Day-7 through Day-10 ICM. Isolation of embryonic stem cell-like colonies was achieved at a higher frequency from ICM isolated from older embryos, but embryonic stem cell-like colonies from older embryos also tended to differentiate spontaneously in culture. Viable porcine chimeras were born after injection of fresh ICM into blastocysts that were transferred to recipients for development to term; no chimeras were born from blastocysts injected with ICM subjected to short-term (1 to 6 d) culture. Germ-cell chimerism was confirmed in one of the chimeras. These results document that undifferentiated cells can be removed from porcine blastocysts, transplanted to other embryos, and contribute to development of normal differentiated tissues, including germ cells. Cells with embryonic stem-like morphology can be isolated in culture from ICM at various embryonic ages, but ICM from young blastocysts (e.g., Day-7 embryos) yield embryonic stem cell-like colonies at lower frequency than do ICM from older blastocysts (e.g., Day-10 embryos).     

Chimeric primates: embryonic stem cells need not apply

In this issue, Tachibana et?al. report the generation of the first chimeras from a nonhuman primate, the rhesus monkey. Unlike mice, rhesus chimeras fail to form when embryonic stem cells are injected into blastocysts. Instead, chimera formation is achieved by aggregation of several four-cell embryos.     

Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras

Ten primary clones of hybrid cells were produced by the fusion of diploid embryonic stem (ES) cells, viz., line E14Tg2aSc4TP6.3 marked by green fluorescent protein (GFP), with diploid embryonic or adult fibroblasts derived from DD/c mice. All the hybrid clones had many characteristics similar to those of ES cells and were positive for GFP. Five hybrid clones having ploidy close to tetraploidy (over 80% of cells had 76–80 chromosomes) were chosen for the generation of chimeras via injection into C57BL blastocysts. These hybrid clones also contained microsatellites marking all ES cell and fibroblast chromosomes judging from microsatellite analysis. Twenty chimeric embryos at 11–13 days post-conception were obtained after injection of hybrid cells derived from two of three clones. Many embryos showed a high content of GFP-positive descendents of the tested hybrid cells. Twenty one adult chimeras were generated by the injection of hybrid cells derived from three clones. The contribution of GFP-labeled hybrid cells was significant and comparable with that of diploid E14Tg2aSc4TP6.3 cells. Cytogenetic and microsatellite analyses of cell cultures derived from chimeric embryos or adults indicated that the initial karyotype of the tested hybrid cells remained stable during the development of the chimeras, i.e., the hybrid cells were mainly responsible for the generation of the chimeras. Thus, ES cell/fibroblast hybrid cells with near-tetraploid karyotype are able to generate chimeras at a high rate, and many adult chimeras contain a high percentage of descendants of the hybrid cells. A. A. Kruglova and E. A. Kizilova contributed equally to this work. This study was financially supported by grants from the Russian Academy of Sciences, Siberian Branch 5.2 and 14.0.     

Developmental incompatibility of human parthenogenetic embryonic stem cells in mouse blastocysts

Human parthenogenetic embryonic stem (pES) cells can be clinically used in the future to avoid immunological rejection. However, the developmental potential of human pES cells remains to be elucidated. In this study, we generated a human pES-enhanced green fluorescent protein (EGFP) cell line (chHES-32-EGFP), which shows pluripotency thus far and maintains stable and robust EGFP expression in the undifferentiated and differentiated states in vivo and in vitro. Using this pES-EGFP cell line, we found that when human pES-EGFP cells were injected into mice blastocysts, EGFP-positive cells progressively decreased with the development of blastocysts in vitro. Only 4 out of 23 embryos (17.4%) contained EGFP-positive cells and all of these embryos exhibited abnormal morphology or delayed development when the chimera blastocysts were implanted into the pseudopregnant recipient mouse uterus. These results raise serious questions regarding the feasibility of the generation of interspecific chimeras between mouse blastocysts and human pES cells.     

远交系小鼠胚胎干细胞系的建立及嵌合鼠的获得

ＥＳ细胞（ＥｍｂｒｙｏｎｉｃＳｔｅｍＣｅｌｌｓ）是来源于小鼠早期胚胎的多潜能干细胞,它可以在体外大量培养。并以单细胞的形式注射到早期胚胎里,发育为嵌合体。到目前为止,通常使用的１２９小鼠品系是来源于近交系（ｉｎｂｒｅｄ）小鼠的胚胎．与之相比,远交系小鼠应当具有较强的生命力和抗病能力。曾有人报道过建成了远交系小鼠胚胎干细胞系,但是尚没有见到获得嵌合鼠的报道。有人甚至认为：由于不同品系小鼠所具有的遗传背景不同,有的小鼠不能建成ＥＳ细胞系。最近,本实验室在这方面做了有益的探索,成功地建成了远交系小鼠胚胎干细胞系,并在这里报导首例用远交系小鼠胚胎干细胞系培育成功嵌合体小鼠。采用源于Ｓｗｉｓｓ小鼠远交群的昆明（ＫＭ）品系小鼠囊胚建成了三个小鼠胚胎干细胞系（ＫＥ１．ＫＥ２．ＫＥ５）。核型正常率均达到７０％以上。自第八代起分批冻存,复苏后,培养至第１２代,消化成单细胞,通过囊胚显微注射,将其注射到６１５品系小鼠胚胎。在幸存的幼鼠中获得了一只来源于ＫＥ１细胞的嵌合鼠（Ｔａｂｌｅ１）．其毛色表现为受体鼠（６１５）的白色中嵌合有供体鼠（ＫＭ）的黑褐色（ＰｌａｔｅＩ－Ａ）．嵌合鼠与受体鼠的杂交后代鼠中仍然出现了受体鼠的毛色类型（     

Production of chimeras by aggregation of embryonic stem cells with diploid or tetraploid mouse embryos

The production of mouse chimeras is a common step in the establishment of genetically modified animal strains. Chimeras also provide a powerful experimental tool for following cell behavior during both prenatal and postnatal development. This protocol outlines a simple and economical technique for the production of large numbers of mouse chimeras using traditional diploid morula<-->diploid embryonic stem (ES) cell aggregations. Additional steps are included to describe the procedures necessary to produce specialized tetraploid chimeras using tetraploid morula<-->diploid ES cell aggregations. This increasingly popular form of chimera produces embryos of nearly complete ES cell derivation that can be used to speed transgenic production or ask developmental questions. Using this protocol, mouse chimeras can be generated and transferred to pseudopregnant surrogate mothers in a 5-d period.     

Identical triplets and twins developed from isolated blastomeres of 8- and 16-cell mouse embryos supported with tetraploid blastomeres

We studied the developmental potential of single blastomeres from early cleavage mouse embryos. Eight- and sixteen-cell diploid mouse embryos were disaggregated and single blastomeres from eight-cell embryos or pairs of sister blastomeres from sixteen-cell embryos were aggregated with 4, 5 or 6 tetraploid blastomeres from 4-cell embryos. Each diploid donor embryo gave eight sister aggregates, which later were manipulated together as one group (set). The aggregates were cultured in vitro until the blastocyst stage, when they were transferred (in sets) to the oviducts of pseudopregnant recipients. Eighteen live foetuses or pups were obtained from the transfer (11.0% of transferred blastocysts) and out of those, eleven developed into fertile adults (one triplet, one pair of twins and four singletons). In all surviving adults, pups and living foetuses, only diploid cells were detected in their organs and tissues as shown by analysis of coat pigmentation and distribution of glucose phosphate isomerase isoforms. In order to explain the observed high rate of mortality of transferred blastocysts, in an accompanying experiment, the diploid and tetraploid blastomeres were labelled with different fluorochromes and then aggregated. These experiments showed the diploid cells to be present not only in the inner cell mass (ICM) but also in the trophectoderm. The low number of diploid cells and the predominance of tetraploid cells in the ICM of chimaeric blastocysts might have been responsible for high postimplantation mortality of our experimental embryos.