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.     

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.     

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.     

Isolated mouse inner cell mass is unable to reconstruct trophectoderm

The ability of ICM to differentiate into TE is still a controversial issue. Many of authors have showed the reconstruction of TE from isolated ICMs. We showed that immunosurgical method is not 100% efficient and that the original TE cells very often remain on the surface of isolated ICMs. We also found that isolated ICM cells cultured in vitro do not express Cdx2, and that the TE is reconstituted from TE cells which have survived immunosurgery. This indicates that very soon after the formation of TE in the blastocyst, the cells of ICM lose the potency to differentiate into trophectoderm.     

Energy metabolism of the inner cell mass and trophectoderm of the mouse blastocyst

Mammalian pre-implantation development culminates in the formation of the blastocyst consisting of two distinct cell lineages, approximately a third of the cells comprise the pluripotent inner cell mass (ICM) and the remainder the differentiated trophectoderm (TE). However, the contribution made by these two cell types to the overall energy metabolism of the intact blastocyst has received relatively little attention. In this study, the metabolism of the intact mouse blastocyst and isolated ICMs were determined in terms of total ATP formation (calculated from oxygen consumption and lactate formation), mitochondrial distribution and amino acid turnover to provide an indication of protein synthesis. The TE consumed significantly more oxygen, produced more ATP and contained a greater number of mitochondria than the ICM. Amino acid turnover was significantly greater (p<0.001) in the TE compared with the ICM. Specifically, there was a significant difference in the utilization of aspartate (p=0.020), glutamate (p=0.024), methionine (p=0.037), and serine (p=0.041) between the cells of the ICM and TE. These data suggest that the TE produces approximately 80% of the ATP generated and is responsible for 90% of amino acid turnover compared with the ICM. The major fate of the energy produced by the TE is likely to be the Na(+), K(+)ATPase (sodium pump enzyme) located on the TE basolateral membrane. In conclusion, the pluripotent cells of the ICM display a relatively quiescent metabolism in comparison with that of the TE.     

Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos

Inner cell mass (ICM) and trophectoderm cell lineages in preimplantation mouse embryos were studied by means of iontophoretic injection of horseradish peroxidase (HRP) as a marker. HRP was injected into single blastomeres at the 2- and 8-cell stages and into single outer blastomeres at the 16-cell and late morula (about 22- to 32-cell) stages. After injection, embryos were either examined immediately for localization of HRP (controls) or they were allowed to develop until the blastocyst stage (1 to 3.5 days of culture) and examined for the distribution of labeled cells. In control embryos, HRP was confined to one or two outer blastomeres. In embryos allowed to develop into blastocysts, HRP-labeled progeny were distributed into patches of cells, showing that there is limited intermingling of cells during preimplantation development. A substantial fraction of injected blastomeres contributed descendants to both ICM and trophectoderm (95, 58, 44, and 35% for injected 2-cell, 8-cell, 16-cell, and late morula stages, respectively). Although more than half of the outer cells injected at 16-cell and late morula stages contributed descendants only to trophectoderm (53 and 63%, respectively), some outer cells contributed also to the ICM lineage even at the late morula stage. Although the mechanism for allocation of outer cells to the inner cell lineage is unknown, our observation of adjacent labeled mural trophectoderm and presumptive endoderm cells implicated polarized cell division. This observation also suggests that mural trophectoderm and presumptive endoderm are derived from common immediate progenitors. These cells appear to separate into inner and outer layers during the fifth cleavage division. Our results demonstrate the usefulness of HRP as a cell lineage marker in mouse embryos and show that the allocation of cells to ICM or trophectoderm begins after the 2-cell stage and continues into late cleavage.     

Do trophectoderm and inner cell mass cells in the mouse blastocyst maintain discrete lineages?

The extent to which trophectoderm (TE) and inner cell mass (ICM) lineages in the mouse blastocyst remain distinct during the period from the commencement of cavitation up until 48 h later in culture was investigated. Fluorescent latex microparticles were used to label exclusively all TE cells in nascent blastocysts and the position of labelled progeny in cultured blastocysts was examined by disaggregation, by serial sectioning and by whole-mount analyses. The results indicate that, in most blastocysts (80-90%), TE and ICM lineages are entirely separate during this period while in the remainder lineage crossing is limited usually to only one or two cells of either tissue.     

A rapid procedure for isolating hemopoietic cell nuclei

A new method for isolating cell nuclei is described which involves freezing and thawing cells in 2% Tween 40, then gentle homogenization to release nuclei, followed by immediate microcentrifugation through 50% sucrose. Purified nuclei were obtained in 3 min and yields of 78-95% were obtained from a variety of human hemopoietic cells. Electron microscope analysis of nuclei obtained from HL60 cells showed that 89% of the nuclei were intact and have an appropriate morphology. A low level of contamination with other organelles was revealed by electron microscopy and by using specific assays for plasma membrane, mitochondria, lysosomes, Golgi membrane, and endoplasmic reticulum (0.5-5.5%). The value of the technique is that nuclear proteins and small metabolites which might be lost by rapid leakage from isolated nuclei and the possibility of biochemical modification of cellular constituents are minimized by using a rapid isolation procedure.     

Induction of pluripotency by injection of mouse trophectoderm cell nuclei into blastocysts following transplantation into enucleated oocytes

Pluripotency of mouse trophectoderm (TE) cells was examined using a nuclear transfer technique. We transferred a TE cell to an enucleated oocyte and cultured the reconstituted oocyte to be blastocyst stage. Then a portion of the inner cell mass (ICM) isolated from the TE-origin blastocyst was injected into the cavity of a fertilized blastocyst to produce a chimeric embryo, which was transferred to a recipient female. Of 319 oocytes reconstituted with TE cells, 263 (82.4%) had a single nucleus (1PN), 3 (0.9%) had 2 nuclei (2PN) and 53 (16.6%) had a nucleus with a polar body (1PN1PB). Although the oocytes with 1PN and 2PN developed to blastocysts (81 of 263, 30.8% and 1 of 3, respectively), only those with 1PN were used to produce chimeric blastocysts. After the transfer of chimeric embryos to recipient females, 7 (28%) of 25 conceptuses analyzed at midgestation showed chimerism. Of those 5 (71%), 6 (86%) and 4 (57%) chimeric conceptuses showed distribution of donor nuclei in the fetus, membrane and placenta, and the distributions were 10 to 65, 10 to 50 and 10 to 15%, respectively. Of the 23 young obtained, 7 (30%; 2 males and 5 females) were coat color chimeras. The contributions of donor nuclei were detected in the brain, lung, heart, liver, kidney, testis, ovary and blood. Each coat-color chimeric mouse was mated with CD-1 male or female mice, but no germ line chimera was obtained. When ICM cells were used as the control nuclear donor, the contribution was equivalent to those of TE cells. In conclusion, pluripotency of mouse TE cells on a somatic line was induced, and chimeric young were obtained using a nuclear transplantation technique.     

Tetraploid embryonic stem cells contribute to the inner cell mass of mouse blastocysts

The demonstration that mouse somatic cells can be reprogrammed following fusion with embryonic stem (ES) cells may provide an alternative to somatic cell nuclear transfer (therapeutic cloning) to generate autologous stem cells. In an attempt to produce cells with an increased pool of reprogramming factors, tetraploid ES cells were produced by polyethylene glycol mediated fusion of two ES cell lines transfected with plasmids carrying puromycin or neomycin resistance cassettes, respectively, followed by double antibiotic selection. Tetraploid ES cells retain properties characteristic of diploid ES cells, including the expression of pluripotent gene markers Oct4 and Rex1. On injection into the testis capsule of severe combined immunodeficient (SCID) mice, tetraploid ES cells are able to form teratomas containing cells representative of all three germ layers. Further, these cells demonstrated the ability to integrate into the inner cell mass of blastocysts. This study indicates that tetraploid ES cells are promising candidates as cytoplasm donors for reprogramming studies.     

Glucose metabolism in the trophectoderm and inner cell mass of the rabbit embryo

The metabolism of glucose in the intact Day-6 and -7 post coitum (p.c.) rabbit blastocyst and in the separated trophectoderm and inner cell mass (ICM) of the Day-7 p.c. embryo was investigated. At Day-6 p.c., glucose traversed the trophectoderm with a half-time of 39 +/- 9.3 min, and was metabolized to CO2 at a rate of 25.5 +/- 1.6 nmol.cm-2.h-1. Neither the Na+ ionophore, amphotericin B, nor cyclic AMP had an effect on glucose metabolism to CO2. Lactate production by the Day-6 blastocyst was largely independent of glucose. At Day-7 p.c. in the intact embryo, CO2 production from glucose significantly decreased to 7.76 +/- 2.8 nmol.cm-2.h-1. Per unit surface area, the metabolism of glucose to CO2 was similar in the separated Day-7 p.c. trophectoderm and ICM. We conclude that the rabbit blastocyst is not highly dependent on glucose, and that the ICM does not utilize glucose as a metabolite to a greater extent than does the trophectoderm, at least in the Day-7 p.c. embryo.     

Trophoblast-specific DNA methylation occurs after the segregation of the trophectoderm and inner cell mass in the mouse periimplantation embryo

The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages, resulting in the formation of the trophectoderm (TE) and inner cell mass (ICM) in blastocysts. Although a lower level of global DNA methylation in the genome of the TE compared with ICM has been suggested, the dynamics of the DNA methylation profile during TE/ICM differentiation has not been elucidated. To address this issue, first we identified tissue-dependent and differentially methylated regions (T-DMRs) between trophoblast stem (TS) and embryonic stem (ES) cells. Most of these TS–ES T-DMRs were also methylated differentially between trophoblast and embryonic tissues of embryonic day (E) 6.5 mouse embryos. Furthermore, we found that the human genomic regions homologous to mouse TS–ES T-DMRs were methylated differentially between human placental tissues and ES cells. Collectively, we defined them as cell-lineage-based T-DMRs between trophoblast and embryonic cell lineages (T–E T-DMRs). Then, we examined TE and ICM cells isolated from mouse E3.5 blastocysts. Interestingly, all T-DMRs examined, including the Elf5, Pou5f1 and Nanog loci, were in the nearly unmethylated status in both TE and ICM and exhibited no differences. The present results suggest that the establishment of DNA methylation profiles specific to each cell lineage follows the first morphological specification. Together with previous reports on asymmetry of histone modifications between TE and ICM, the results of the current study imply that histone modifications function as landmarks for setting up cell-lineage-specific differential DNA methylation profiles.     

Rabbit blastocyst: Allocation of cells to the inner cell mass and trophectoderm

The proportion of total cells in the blastocyst allocated to the inner cell mass (ICM) and trophectoderm (TE) is important for future development and may be a sensitive indicator to evaluate culture conditions. The number of cells and their distribution within the two primary cell lineages were determined for the rabbit embryo developing in vivo after superovulation or nonsuperovulation or embryo transfer and compared with embryos developing in vitro. Comparisons were made with cultured embryos or embryos grown in vivo until 3.5, 4.0, and 4.5 days of age. Embryos from superovulated rabbits developed in vivo for 3.5, 4.0, and 4.5 days, respectively, had 361, 758, and 902 total cells (P<0.05), and in nonsuperovulated rabbits 130, 414, and 905 total cells (P<0.05), with increasing proportions of ICM cells over time (P<0.05). One-cell embryos recovered from superovulated females and transferred to nonsuperovulated recipients developed more slowly with 70, 299, and 550 total cells after 3.5, 4.0, and 4.5 days of culture (P<0.05), respectively. The proportion of ICM cells increased with age of the embryo. Corresponding values for one-cell embryos cultured in vitro resulted in 70, 299, and 550 total cells (P<0.05). However, in vitro culture of morula-stage embryos in the presence of fetal bovine serum for 24 hr did not delay growth. In addition, the proportions of ICM/total cells were 0.17, 0.25, and 0.29 for embryos developing in vitro at 3.5, 4.0, and 4.5 days, respectively, similar to those for embryos developing in vivo at each of the three recovery times. These data establish for the first time the number and proportion of cells allocated to the ICM of the rabbit embryo developing in vivo or under defined conditions in vitro. © 1995 Wiley-Liss, Inc.     

Maintenance of the inner cell mass in human blastocysts from fragmented embryos

The degree of fragmentation during early cleavage is universally used as an indicator of embryo quality during human in vitro fertilization treatment. Extensive fragmentation has been associated with reduced blastocyst formation and implantation. We examined the relationship between early fragmentation and subsequent allocation of cells to the trophectoderm and inner cell mass in the human blastocyst. We retrospectively analyzed data from 363 monospermic human embryos that exhibited varying degrees of fragmentation on Day 2. Embryos were cultured from Day 2 to Day 6 in Earle balanced salt solution with 1 mM glucose and human serum albumin. Rates of development and blastocyst formation were measured. The number of cells in the trophectoderm and inner cell mass and the incidence of apoptosis were assessed following differential labeling with polynucleotide-specific fluorochromes. Increasing fragmentation resulted in reduced blastocyst formation and lower blastocyst cell numbers. For minimal and moderate levels of fragmentation, the reduction in cell numbers was confined largely to the trophectoderm and a steady number of inner cell mass cells was maintained. However, with extensive fragmentation of more than 25%, cell numbers in both lineages were reduced in the few embryos that formed blastocysts. Apoptotic nuclei were present in both the trophectoderm and inner cell mass, with the lowest incidence in blastocysts that had developed from embryos with minor (5-10%) fragmentation. Paradoxically, higher levels of apoptosis were seen in embryos of excellent morphology, suggesting a possible role in regulation of cell number.     

The developmental potential of the inner cell mass of blastocysts that were derived from mouse ES cells using nuclear transfer technology

The present study examined the causes of the low developmental potential of enucleated oocytes that have received ES cells and consequent postnatal death of the young. The inner cell masses (ICM) of nuclear-transferred blastocysts or diploid blastocysts were injected into tetraploid blastocysts (group B) or nuclear-transferred tetraploid blastocysts (group C), respectively. The developmental potential of these groups was compared with tetraploid blastocysts injected with ICM of diploid blastocysts (group A). The potential of reconstituted blastocysts to develop into live young in group B increased slightly (5%) but was significantly lower than that in group A (45%). The rate of postnatal death of young in group B did not decrease. The implantation rate of reconstituted blastocysts in group C was very low and no live fetuses were obtained. The results of the present study indicate that the inferior potential of both ICM and trophectoderm cells of nuclear-transferred blastocysts underlies the low developmental rate of nuclear-transferred oocytes receiving ES cells and the higher rate of postnatal death of ES cell-derived young.     

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.     

Potential of isolated mouse inner cell masses to form trophectoderm derivatives in vivo

Recent in vitro experiments on immunosurgically isolated mouse inner cell masses (ICMs) have suggested that some ICM cells may retain the potential to form trophectoderm after initial blastocyst formation. These experiments relied almost solely on in vitro morphology for identification of trophectoderm derivatives and provided no proof that the putative trophectoderm cells were capable of functioning in utero. We present clear in vivo evidence that at least some cells in ICMs isolated from early blastocysts do retain the potential to form postimplantation trophectoderm derivatives. Early ICMs occasionally contributed to trophoblast fractions in ICM/morula aggregation chimeras. More strikingly, when aggregated with each other, these ICMs were capable of implanting in the uterus, a property of trophectoderm cells alone. Indeed, some aggregates reconstituted complete egg cylinders. However, ICMs isolated from later blastocysts rarely produced in vivo trophoblast, and it appears that the ability to form trophectoderm is lost around the 16–19 cell ICM stage. These results are discussed in relation to changing patterns of gene activity in early development.