Interspecies chimera between primate embryonic stem cells and mouse embryos: monkey ESCs engraft into mouse embryos, but not post-implantation fetuses

Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspring has yet to be demonstrated in human or nonhuman primates (NHPs). Here, rhesus and baboons ESCs were investigated in interspecific mouse chimera generated by aggregation or blastocyst injection. Aggregation chimera produced mouse blastocysts with GFP-nhpESCs at the inner cell mass (ICM), and embryo transfers (ETs) generated dimly-fluorescencing abnormal fetuses. Direct injection of GFP-nhpESCs into blastocysts produced normal non-GFP-fluorescencing fetuses. Injected chimera showed >70% loss of GFP-nhpESCs after 21 h culture. Outgrowths of all chimeric blastocysts established distinct but separate mouse- and NHP-ESC colonies. Extensive endogenous autofluorescence compromised anti-GFP detection and PCR analysis did not detect nhpESCs in fetuses. NhpESCs localize to the ICM in chimera and generate pregnancies. Because primate ESCs do not engraft post-implantation, and also because endogenous autofluorescence results in misleading positive signals, interspecific chimera assays for pluripotency with primate stem cells is unreliable with the currently available ESCs. Testing primate ESCs reprogrammed into even more na?ve states in these inter-specific chimera assays will be an important future endeavor.     

Production of mouse by inter-strain inner cell mass replacement

The developmental ability of reconstructed blastocysts from C57BL/6 strain mouse inner cell masses (ICMs) and Kunming strain mouse trophoblasts was assessed. The procedure of ICM replacement was as follows: C57BL/6 ICMs were separated from the blastocysts using immunosurgery. A slit was made in the zona pellucida of a Kunming blastocyst to allow its ICM to extrude. The C57BL/6 ICM was injected into the Kunming blastocoele, and the extruded Kunming ICM was cut off. The reconstructed blastocysts were able to re-expand (77%) and hatch (27.3%) in vitro. A total of 64 reconstructed blastocysts and 124 Kunming blastocysts were co-transferred into the uteri of 11 pseudopregnant Kunming mice, and an ICM replacement offspring was born. The results indicate that reconstructed embryos obtained by inter-strain ICM replacement have the ability to develop to term. This technique may provide a method to solve the pregnancy failure in interspecific cloning.     

Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro

The in vitro viability of polyspermic pig eggs was investigated. Immature oocytes were matured and fertilized in vitro. Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g for 10 min and individually classified into two (2PN)- and poly-pronuclear (PPN, 3 or 4 pronuclei) eggs. The classified eggs were cultured in vitro or in vivo. Nuclei numbers of inner cell mass (ICM) and trophectoderm (TE) were compared between 2PN- and PPN-derived blastocysts. The frequency of development in vitro of 2PN and PPN eggs to the blastocyst stage was 53.6% and 40.7%, respectively. The mean number (8.2 +/- 0.7, n = 48) of ICM nuclei of 2PN-derived blastocysts was higher than that (4.2 +/- 0.8, n = 37) of PPN-derived blastocysts (p < 0.001), whereas there was no difference (p > 0.05) in mean numbers of total (46.7 +/- 3.4 vs. 39. 9 +/- 3.9) and TE nuclei (38.5 +/- 2.9 vs. 35.7 +/- 3.3) between the two groups. Development of 2PN and PPN eggs cultured in vivo to the blastocyst stage was 33.3% and 27.4%, respectively. The numbers of ICM and TE nuclei of these embryos cultured in vivo showed a pattern similar to that for the in vitro-produced blastocysts. Additionally, fetuses were obtained on Day 21 from both the 2PN and the PPN groups. This suggests that polyspermic pig embryos develop to the blastocyst stage and beyond, although showing a smaller ICM cell number as compared to normal embryos.     

Relationship between timing of development,morula morphology,and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos

Noninvasive measurements of bovine embryo quality, such as timing of cleavage, morula morphology, blastocyst formation, and hatching ability, were linked with the number of inner cell mass (ICM) cells and trophectoderm (TE) cells of the resulting embryos. First, it was confirmed that fast-cleaving embryos proved to have significantly higher chances to reach advanced developmental stages vs. intermediate and slow cleavers (P = 0.01). They also showed significantly less fragmentation at the morula stage, implying the presence of more excellent morulae among fast-cleaving embryos (P < 0.05). Second, the quality of hatched blastocysts, resulting from morulae of different morphological grades, was examined by differential staining. The total cell and ICM cell numbers were significantly lower for hatched blastocysts developed from poor morulae compared to hatched blastocysts developed from excellent, good, or fair morulae. However, hatched blastocysts with <10 ICM cells were seen in embryos belonging to all four morphological scores. Finally, it was found that timing of first cleavage was not significantly correlated with timing of blastocyst formation or with cell number of blastocysts. Timing of blastocyst formation, however, was significantly correlated with cell number: day 8 blastocysts had significantly lower total cell and ICM cell numbers than day 6 and day 7 blastocysts (P < 0.001). These results suggest that the quality of in vitro-produced bovine embryos is very variable and cannot be linked with a single criterion such as embryo morphology and/or hatching ability. Timing of blastocyst formation was the most valuable criterion with regard to embryonic differentiation. Mol. Reprod. Dev. 47:47–56, 1997. © 1997 Wiley-Liss, Inc.     

Beneficial effects of dithiothreitol on relative levels of glutathione S-transferase activity and thiols in oocytes, and cell number, DNA fragmentation and allocation at the blastocyst stage in the mouse

We analyzed the effect of in vitro aging of mouse oocytes in the presence of dithiothreitol (DTT) on relative levels of glutathione S-transferase (GST) activity and thiols in oocytes, and cell number, DNA fragmentation and cellular allocation to the inner cell mass (ICM) and trophectoderm (TE) lineage at the blastocyst stage. Ovulated oocytes from gonadotropin primed hybrid female mice of 6-8 weeks of age were aged in vitro in the presence of 0, 5, 50, or 500 microM DTT for 6 hr prior to insemination. Relative levels of GST activity and thiols in oocytes were determined by confocal laser scanning microscopy, DNA fragmentation using a single-step TUNEL method, and cell allocation to the ICM and TE lineage by blastocyst staining with propidium iodide and Hoechst 33258. Non-aged oocytes exhibited higher relative levels of GST activity and thiols when compared to oocytes aged in the presence of 0, 5, and 50 microM DTT. Day 5 blastocysts from the 5, 50, and 500 microM DTT groups exhibited higher total number of cells, number of ICM cells, and ICM/TE ratio, but lower percentage of number of nuclei with DNA fragmentation/number of ICM cells than blastocyst from the 0 microM DTT group. These data show that DTT counteracts the negative effects of a post-ovulatory aging of mouse oocytes in vitro on relative levels of GST activity and thiols in oocytes, and percentage of number of nuclei with DNA fragmentation/number of ICM cells, total number of cells, number of ICM cells and ICM/TE ratio in Day 5 blastocysts.     

Identification of specific gene sequences in preimplantation embryos by genomic amplification: detection of a transgene

Endogenous and foreign DNA sequences can be detected in an extremely small number of cells via sequence amplification in vitro. The polymerase chain reaction (PCR) technique applied in multiple cycles allows the amplification of specific short regions of the genome to levels that can be detected by DNA blotting techniques. Cow and mouse blastocysts were analyzed by PCR for the presence of an endogenous singlecopy gene or an integrated foreign gene. The endogenous single-copy gene encoding the beta chain of bovine luteinizing hormone was detectable in cow blastocysts and in purified bovine genomic DNA representing as few as 25 cells. To determine whether exogenous genes (transgenes) can be detected in preimplantation embryos, transgenic male mice hemizygous for the prokaryotic gene encoding neomycin resistance were bred to nontransgenic females, and the resulting blastocysts were analyzed. The neo gene was detected in approximately half of the embryos. The capability to identify specific gene sequences in a limited number of embryonic cells affords investigators the opportunity to study genetics in early development.     

Enhancement of radiation effects by mercury in early postimplantation mouse embryos in vitro

Summary Two-cell mouse embryos were X-irradiated (1 Gy) and immediately thereafter exposed to mercuric chloride (3 µM) up to the blastocyst stage. When combined treatment started shortly (about 1 to 2 h) before mitosis to the four-cell stage, blastocyst formation, hatching of blastocysts, trophoblast outgrowth and ICM formation were impaired stronger than expected from the addition of the single effects. The enhancement of risk was maximal for hatching of blastocysts and no further increase was observed for trophoblast outgrowth and ICM formation. When exposure of embryos to X-rays and mercury began about 5 to 6 h before mitosis to the four-cell stage, only additive effects were obtained for the endpoints mentioned above.     

Morphological development,cell number,and allocation of cells to trophectoderm and inner cell mass of in vitro fertilized and parthenogenetically developed buffalo embryos: The effect of IGF-I

The morphology and number of cells in the trophectoderm (TE) and inner cell mass (ICM) of buffalo blastocysts derived from in vitro fertilization and cultured in the presence or absence of insulin-like growth factor-I (IGF-I) were analyzed by differential fluorochrome staining technique. The total cell number (TCN), TE number, and ICM cell number were significantly higher in blastocysts developed in vitro in the presence of IGF-I as compared to blastocysts developed without IGF-I (P < 0.01). It was observed that the buffalo blastocyst took 5–9 days postfertilization to develop in vitro. In order to correlate the time required for blastocyst development and the allocation of cells to TE and ICM, blastocysts were designated as fast (developing on or before day 7) or slow (developing after day 7). The TCN, TE, and ICM cells of fast-developing blastocysts cultured in the presence of IGF-I were significantly higher than slow-developing blastocysts (P < 0.01). The blastocysts developed on day 6 had a mean total cell number 118.6 ± 21.4, which significantly decreased to 85.6 ± 17.4, 62.0 ± 14.5, and 17.0 ± 4.0 on days 7, 8, and 9, respectively (P < 0.05). Normal development of buffalo embryo showed that, on average, embryos reached compact morula stage at the earliest between days 4.5–5.5. Blastocysts developed, at the earliest, between days 5.0–6.0, and it took them, on average, 6.5 days to hatch from the zona pellucida. TCN, TE, and ICM increased three times from morula to blastocyst; however, the proportion of ICM to TCN remained the same, in both embryonic stages. TE approximately doubled in hatched blastocysts, as compared to unhatched blastocysts (P < 0.05). However, ICM cells were decreased. The time required for development of parthenogenetic blastocysts was observed to be greater as compared to in vitro fertilized (IVF) blastocysts. The total cell number of parthenogenetic blastocysts was 100.8 ± 11.3, including 59.2 ± 8.4 cells of TE and 42.1 ± 6.9 cells of ICM. © 1996 Wiley-Liss, Inc.     

Transgene detection during early murine embryonic development after pronuclear microinjection

The polymerase chain reaction (PCR) technique was used to detect a whey acidic protein (WAP) gene and transgene presence in mouse ova cultured to various stages of development after pronuclear microinjection at the one-cell stage. The PCR technique detected an endogenous 442 bp WAP DNA sequence in 78% of one-cell, 88% of two-cell and 94% of four-cell ova, and in 95% of morulae and 97% of blastocysts. The heterologous WAP-human protein C transgene was detected in 88% of one-cell, 88% of two-cell and 44% of four-cell ova, and in 40% of morulae and 29% of blastocysts. For comparison, the integration frequency for transgenic mouse production using the same DNA construct was 22%. After five days ofin vitro culture, embryos that were either developmentally arrested or fragmented were tested for the presence of the transgene. The injected construct was detected in 83% of arrested one-cell, 85% of arrested two-cell, and 85% of fragmented ova. In culture, only 28% of zygotes microinjected with DNA developed to the blastocyst stage compared to 74% of noninjected zygotes, while 63% of zygotes developed to the blastocyst stage after injection of buffer alone. Pronuclear injection of the transgene at concentrations of 1.5, 15 and 50 g ml^–1 resulted in 28, 11 and 9% development to blastocysts and 29, 86 and 88% transgene detection, respectively. Transgene detection was 85, 96 and 97% in degenerate embryos at the respective doses of DNA. These data show that pronuclear microinjection of the transgene is detrimental to subsequent embryonic development. Also, unintegrated copies of the transgene probably exist at least until the blastocyst stage, and thereafter are degraded to the extent that they can no longer be detected by PCR.     

A paucity of structural integrity in cloned porcine blastocysts produced in vitro

The structural integrity of blastocyst stage embryos, consisting of the inner cell mass (ICM) and trophectoderm (TE) cells, is a prerequisite for normal development after implantation in mammals. In this study, allocation of nuclear transfer (NT)-derived porcine blastocysts to the ICM and to the TE cells was examined and compared with IVF- and in vivo-derived embryos. NT-derived embryos had a lower developmental competence to the blastocyst stage than IVF-derived embryos (P < 0.05). Total cell number of NT-derived blastocysts was inferior to that of IVF-derived embryos (P < 0.05), although no difference was detected between the two groups in the ratio of ICM to total cells. However, in vivo-derived blastocysts had a higher proportion of ICM to total cells compared with in vitro-produced embryos (P < 0.01). To investigate what proportions of in vitro-produced porcine embryos represent normal structural integrity, differentially-stained blastocysts were individually classified into three presumptive groups (I: <20%; II: 20-40%; III: >40%) according to the ratio of ICM to total cells. Low proportions of NT- (12.5%, 7/56) and IVF-derived blastocysts (15.8%, 9/57) were assigned to Group II, presumptively having a normal range of structural integrity, whereas, almost all in vivo-derived embryos (97.5%, 39/40) were allocated to Group II. In conclusion, limited structural integrity may lead to the poor survival to term of NT- or IVF-derived porcine embryos produced in vitro.     

Effect of chemically defined culture medium supplemented with beta-mercaptoethanol and amino acids on implantation and development of different stage in vivo- or in vitro-derived mouse embryos

In vitro culture (IVC) systems are required for many biotechnological and assisted reproductive technologies and the researchers have been modifying in vitro embryo culture conditions to reach the comparable efficiencies provided in vivo. In the present study, the effects of beta-mercaptoethanol (Beta-ME) and amino acids (AA) on the development of mouse embryos obtained in vivo or in vitro at different stages were investigated. Chemically defined potassium simplex optimized medium (KSOM) was used as basic culture medium and six experimental groups were established and by supplementation of Beta-ME and AA into KSOM media. The quality of blastocysts was evaluated by counting the cells and determining the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells. In addition, embryo transfer (ET) was performed to investigate the rate of implantation and live fetuses. The results obtained in the present study demonstrated that the combined treatment of Beta-ME and AA to 1-cell stage embryos not only enhanced in vitro development to the blastocyst stage but also improved both the number of blastocysts cells and live fetuses.     

A comparative investigation on the potency of cells from the inner cell mass and trophectoderm of mouse blastocysts to produce chimeras

The ability of trophectoderm (TE) cells to produce chimeric mice (pluripotency) was compared with that of inner cell mass (ICM) cells. TE and ICM cells of blastocysts and hatching or hatched blastocysts derived from albino mice (CD-1, Gpi-1a/a) were aggregated with zona cut 8- to 16-cell stage embryos or injected into the blastocoele from non-albino mice (C57BL/6 x C3H/He, Gpi-1b/b). After transfer to pseudopregnant female mice, the contribution of the donor cells was examined by glucose phosphate isomerase (GPI) analysis of embryos, membrane and placenta at mid-gestation (Day 10.5 and 12.5) or by the coat color of newborn mice. In contrast to ICM cells, there was no contribution of TE cells in the conceptuses and no coat color chimeric young were obtained. After pre-labeling of TE cells with fluorescent latex microparticles, they were aggregated with embryos and the allocation of TE cells at the compacted morula and blastocyst stages was observed under a fluorescent microscope. Although the TE cells were observed attached onto the surface of the embryos at morula and blastocyst stages, unlike the ICM cells, they were not positively incorporated into the embryos. Thus, the pluripotency of TE cells from mouse blastocysts was not induced by the aggregation and injection methods.     

Cell fate in the polar trophectoderm of mouse blastocysts as studied by microinjection of cell lineage tracers

Horseradish peroxidase (HRP), together with Fast Green or rhodamine-conjugated dextran (RDX), was used as an intracellular lineage tracer to determine cell fate in the polar trophectoderm of 3.5-day-old mouse embryos. In HRP-injected midstage (approximately 39-cell) and expanded (approximately 65-cell) blastocysts incubated for 24 hr, the central polar trophectoderm cell was displaced from the embryonic pole an average of 20 micron (5% of blastocyst circumference) and 29 micron (6% of blastocyst circumference), respectively. Expanded blastocysts injected with HRP + Fast Green and incubated for 24 hr or with HRP + RDX and incubated for 48 hr showed a displacement of 24 micron (4% of blastocyst circumference) and 88 micron (14% of blastocyst circumference), respectively. Up to 10 HRP-positive trophectoderm cells were observed among embryos incubated for 48 hr, indicating that in those cases, the labeled progenitor cells had divided at least three times. Our observations show that the central polar trophectoderm cell divides in the plane of the trophectoderm in expanded blastocysts and, along with its descendants, is displaced toward the mural trophectoderm. The systematic tandem displacement of labeled cells and their descendants toward the abembryonic pole suggests the presence of a proliferative area at the embryonic pole of the blastocyst. Large shifts in inner cell mass (ICM) position in relation to the trophectoderm do not occur during blastocyst expansion. Furthermore, random movements within the polar trophectoderm population do not account for the replacement of labeled cells by unlabeled polar trophectoderm cells. Rather, we propose the hypothesis that the ICM contributes these replacement cells to the polar trophectoderm during blastocyst expansion.     

Postimplantation development of mitomycin C-treated mouse blastocysts

Treatment of morula-stage mouse embryos with mitomycin C (0.004-0.5 microgram/ml) in vitro resulted in a decrease in the number of inner cell mass (ICM) cells at the blastocyst stage. The trophectoderm population was reduced only at the highest dosage (0.5 microgram/ml) tested. Postblastocyst development in vitro was retarded: Fewer embryos formed trophoblastic outgrowth, and the ICM was poorly developed. The embryo transfer experiments demonstrated that a reduction in ICM cell numbers diminished the potential of embryogenesis. The presence of a sufficient number of trophoblasts and ICM cells in the blastocyst is therefore a prerequisite for successful implantation and embryogenesis. The mitomycin-treated blastocysts with only 70% of normal ICM cells developed to egg cylinders that were about half normal size, but by days 12-14 the body size of the surviving embryo was similar to that of the control embryo. Morphogenesis was retarded during the early organogenesis stages, but only a slight delay was seen in the treated embryo on day 12. Such observation strongly suggests that a restorative phase of growth and morphogenesis has occurred during the immediate postimplantation period.     

A quantitative analysis of cell allocation to trophectoderm and inner cell mass in the mouse blastocyst

The allocation of cells to the trophectoderm and inner cell mass (ICM) in the mouse blastocyst has been examined by labelling early morulae (16-cell stage) with the short-term cell lineage marker yellow-green fluorescent latex (FL) microparticles. FL is endocytosed exclusively into the outside polar cell population and remains autonomous to the progeny of these blastomeres. Rhodamine-concanavalin A was used as a contemporary marker for outside cells in FL-labelled control (16-cell stage) and cultured (approximately 32- to 64-cell stage) embryos, immediately prior to the disaggregation and analysis of cell labelling patterns. By this technique, the ratio of outside to inside cell numbers in 16-cell embryos was shown to vary considerably between embryos (mean 10.8:5.2; range 9:7 to 14:2). In cultured embryos, the trophectoderm was derived almost exclusively (over 99% cells) from outside polar 16-cell blastomeres. The origin of the ICM varied between embryos; on average, most cells (75%) were descended from inside nonpolar blastomeres with the remainder derived from the outside polar lineage, presumably by differentiative cleavage. In blastocysts examined by serial sectioning, polar-derived ICM cells were localised mainly in association with trophectoderm and were absent from the ICM core. In nascent blastocysts with exactly 32 cells an inverse relationship was found between the proportion of the ICM descended from the polar lineage and the deduced size of the inside 16-cell population. From these results, it is concluded that interembryonic variation in the outside to inside cell number ratio in 16-cell morulae is compensated by the extent of polar 16-cell allocation to the ICM at the next division, thereby regulating the trophectoderm to ICM cell number ratio in early blastocysts.     

Effects of freezing of bovine preimplantation embryos derived from oocytes fertilized in vitro on survival of their inner cell mass cells

The morphology of the inner cell mass (ICM) cells and the proportion of dead ICM cells in frozen-thawed bovine preimplantation embryos were investigated by differential fluorochrome staining. Embryos at the blastocyst stage of development were frozen and thawed by two different techniques (three-step and one-step) in two different basic salt solutions (PBS and TCM 199) containing 1.36M glycerol. After thawing and glycerol removal, embryos were co-cultured in a cumulus cells monolayer in TCM 199 for 48 hr (morula) or 24 hr (blastocysts). Differential cell counts of the ICM and trophectoderm were then done using differential fluorochrome staining. Overall, there was no significant difference in the viability of embryos frozen in the two basic salt solutions. Low proportions of dead ICM cells were observed in embryos frozen at the morula stage in both PBS (19.1%) or TCM 199 (18.0%). However, blastocyst stage embryos frozen by the three-step technique had a higher (P < 0.05) proportion of dead ICM cells in TCM 199 (37.7%) than in PBS (18.2%). Blastocysts frozen by the one-step technique had a higher (P < 0.05) proportion of dead ICM cells (42.2%) than those frozen by the three-step technique (18.2%), regardless of basic salt solutions. Results indicate that freezing and thawing damages ICM cells in morphologically normal embryos and that the degree of damage depended on the basic salt solution and the freezing method. © 1994 Wiley-Liss, Inc.     

Role of the extracellular matrix protein thrombospondin in the early development of the mouse embryo

The distribution of the extracellular matrix protein thrombospondin (TSP) in cleavage to egg cylinder staged mouse embryos and its role in trophoblast outgrowth from cultured blastocysts were examined. TSP was present within the cytoplasm of unfertilized eggs; in fertilized one- to four-cell embryos; by the eight-cell stage, TSP was also densely deposited at cell-cell borders. In the blastocyst, although TSP was present in all three cell types; trophectoderm, endoderm, and inner cell mass (ICM), it was enriched in the ICM and at the surface of trophectoderm cells. Hatched blastocysts grown on matrix-coated coverslips formed extensive trophoblast outgrowths on TSP, grew slightly less avidly on laminin, or on a 140-kD fragment of TSP containing its COOH terminus and putative cell binding domains. There was little outgrowth on the NH2 terminus heparin-binding domain. Addition of anti-TSP antibodies (but not GRGDS) to blastocysts growing on TSP strikingly inhibited outgrowth. Consistent with its early appearance and presence in trophoblast cells during implantation, TSP may play an important role in the early events involved in mammalian embryogenesis.     

Aberrant allocations of inner cell mass and trophectoderm cells in bovine nuclear transfer blastocysts

Abortions of nuclear transfer (NT) embryos are mainly due to insufficient placentation. We hypothesized that the primary cause might be the aberrant allocations of two different cell lineages of the blastocyst stage embryos, the inner cell mass (ICM) and the trophectoderm (TE) cells. The potential for development of NT embryos to blastocysts was similar to that for in vitro fertilized (IVF) embryos. No difference in the total cell number was detected between NT and IVF blastocysts, but both types of embryos had fewer total cells than did in vivo-derived embryos (P < 0.05). The NT blastocysts showed a higher ratio of ICM:total cells than did IVF or in vivo-derived embryos (P < 0.05). Individual blastocysts were assigned to four subgroups (I: <20%, II: 20-40%, III: 40-60%, IV: >60%) according to the ratio of ICM:total cells. Most NT blastocysts were placed in groups III and IV, whereas most IVF and in vivo-derived blastocysts were distributed in group II. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocations of NT embryos to the ICM and TE cells during early development.     

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.