141 A NOVEL METHOD FOR PURIFICATION OF INNER CELL MASS AND TROPHECTODERM CELLS FROM BOVINE BLASTOCYSTS USING MAGNETIC ACTIVATED CELL SORTING

2011 ◽  
Vol 23 (1) ◽  
pp. 174
Author(s):  
M. Ozawa ◽  
P. J. Hansen
Keyword(s):  
Cell Sorting ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Expression Level ◽  
Hoechst 33342 ◽  
Mammalian Embryo ◽  
Positive Fraction ◽  
Relative Expression Level ◽  
Single Blastomeres ◽  
Magnetic Activated Cell Sorting

The first distinct lineage differentiation in the mammalian embryo occurs at the blastocyst stage when blastomeres are segregated into inner cell mass (ICM) or trophectoderm (TE). Obtaining purified TE or ICM can be useful for understanding regulation of early development and differentiation. Although several methods have been reported to separate TE and ICM (e.g. immunosurgery, mechanical dissection using a micromanipulator, or manual selection following trypsinization), limitations exist with these methods. Here, we describe a simple and effective method to sort cells of the blastocyst using magnetic activated cell sorting (MACS) following disaggregation of the blastocyst into single cells using trypsin. Bovine blastocysts were produced in vitro and the zona pellucida removed with a short exposure to acidic Tyrode’s solution. Zona-free blastocysts were incubated with concanavalin A conjugated to fluorescein isothiocyanate (FITC) to label the outer layer of the blastocyst. The blastocysts were then exposed to Hoechst 33342 to label nuclei of all blastomeres. The blastocysts were treated with 0.05% (wt/vol) trypsin, and then disaggregated into single blastomeres by repeating pipetting using a finely drawn, flame-polished mouth micropipette. Single blastomeres were incubated with magnetic microbeads conjugated to anti-FITC and subjected to MACS separation. A fraction of sorted cells was observed under a fluorescence microscope. The remainder were subjected to mRNA extraction, and NANOG (ICM marker) and CDX2 (TE marker) mRNA were quantified by quantitative PCR. After disaggregation of the blastocyst, 2 types of single blastomeres were observed: cells that were positive for both FITC and Hoechst 33342 (TE cells) and cells that were negative for FITC but positive for Hoechst 33342 (ICM cells). Before MACS, about two-thirds of the disaggregated blastomeres labelled with Hoechst 33342 were also labelled with FITC, while one-third were FITC negative. After MACS, the percent of dual-labelled cells in the FITC positive fraction was 91.2%, whereas the incidence of dual-labelled cells in the FITC negative fraction was only 7.8 ± 3.0%. A total of 11.5 μg of RNA per blastocyst was recovered from cells isolated by MACS. This represents 80% of the RNA present in intact blastocysts and suggests a high rate of recovery of blastomeres during the purification process. Furthermore, relative expression level of NANOG was lower in the FITC-positive fraction than in the FITC-negative fraction (0.30 ± 0.05 v. 3.1 ± 0.6, respectively, relative to gene expression level in whole blastocysts). Conversely, the relative expression level of CDX2 was higher in the FITC-positive fraction than in the FITC-negative fraction (3.2 ± 0.09 v. 0.30 ± 0.9, respectively). Results indicate that highly purified TE cells or ICM cells can be collected using MACS. This simple method can be used to study differentiation of the mammalian embryo as well as to prepare embryonic cells of specific lineages for cell therapy. Research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2009-65203-05732 from the USDA NIFA.

scholarly journals Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marino Maemura ◽  
Hiroaki Taketsuru ◽  
Yuki Nakajima ◽  
Ruiqi Shao ◽  
Ayaka Kakihara ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Primitive Endoderm ◽  
Cell Stage ◽  
Supportive Environment ◽  
Mouse Zygotes ◽  
Single Blastomeres ◽  
Multicellular Organisms

AbstractIn multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

202 Targeting Galactosyl-α-1,3-Galactose (αGal) Epitopes for Multi-Species Embryo Immunosurgery

2018 ◽  
Vol 30 (1) ◽  
pp. 241
Author(s):  
M. Kurome ◽  
A. Baehr ◽  
K. Simmet ◽  
B. Kessler ◽  
E. Jemiller ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Fluorescein Isothiocyanate ◽  
Transcriptome Profiling ◽  
Hoechst 33342 ◽  
Experimental Variation ◽  
Reliable Source ◽  
German Research ◽  
Almost All ◽  
Pig Serum

Immunosurgical isolation of the inner cell mass (ICM) from blastocysts is based on complement-mediated lysis of antibody-coated trophectoderm (TE) cells. Conventionally, anti-species antisera, containing antibodies against multiple undefined TE cell epitopes, have been used as antibody source. We previously generated α-1,3-galactosyltransferase deficient (GTKO) pigs to prevent hyper-acute rejection of pig-to-primate xenotransplants. Because GTKO pigs lack galactosyl-α-1,3-galactose (αGal) but are exposed to this antigen (e.g. αGal on gut bacteria), they are expected to produce anti-αGal antibodies. In this study, we examined whether serum from GTKO pigs can be used as a novel antibody source for embryo immunosurgery. First, the presence of αGal epitopes in mouse (E3.5), rabbit (Day 4), pig (Day 6–7), and bovine (Day 7–8) blastocysts was examined by staining with fluorescein isothiocyanate (FITC)-conjugated BSI-B4 lectin (Sigma, St. Louis, MO, USA) that binds αGal. Expression of αGal epitopes on the surface of TE cells was detected in blastocysts of all examined species. Next, pig blastocysts were incubated with a medium containing GTKO pig serum. Swollen TE cells were observed in some of the blastocysts already after 2 min and, after 10 min, almost all TE cells of these blastocysts were completely destroyed. No lysis was recorded when the same experiment was done with wild-type pig serum, suggesting the presence of sufficient quantities of anti-αGal antibodies in GTKO serum to coat the TE cells and induce their complement-mediated lysis. Finally, GTKO serum was systematically tested for immunosurgery. Zona-free blastocysts of the species mentioned above were incubated with heat-inactivated GTKO pig serum for 1 h at 38°C. After washing, the blastocysts were labelled with Hoechst 33342 and TE was stained with FITC-conjugated concanavalin A (ConA) to distinguish the ICM from TE cells. Eventually, the blastocysts were individually incubated in complement solution for 30 to 40 min. Complement-mediated lysis of TE cells was efficiently induced in mouse, rabbit, pig, and bovine blastocysts (10/10, 7/7, 10/10, and 5/6, respectively), and intact ICM were successfully recovered from all species (100, 100, 60, and 80%, respectively). Double fluorescent staining with Hoechst 33342 and ConA clearly showed that the majority of isolated ICM was not contaminated with TE cells. Our study demonstrates that GTKO pig serum is a reliable source of antibodies targeting the αGal epitope of TE cells. Major advantages of using GTKO serum for embryo immunosurgery are (1) that it can be produced easily in large batches, thus reducing experimental variation; and (2) that it reacts with a large number of different species, except for humans, apes, and old world monkeys that lack αGal epitopes. Interesting applications include the preparation of TE and ICM for transcriptome profiling or chimeric embryo complementation experiments. This work is supported by the German Research Council (TR-CRC 127).

scholarly journals Multiscale analysis of single and double maternal-zygotic Myh9 and Myh10 mutants during mouse preimplantation development

2020 ◽  
Author(s):  
Markus Frederik Schliffka ◽  
Anna-Francesca Tortorelli ◽  
Özge Özgüç ◽  
Ludmilla de Plater ◽  
Oliver Polzer ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Preimplantation Development ◽  
Mammalian Development ◽  
Mammalian Embryo ◽  
Fate Specification ◽  
Heavy Chains ◽  
A Cell ◽  
The Impact

AbstractDuring the first days of mammalian development, the embryo forms the blastocyst, the structure responsible for implanting the mammalian embryo. Consisting of an epithelium enveloping the pluripotent inner cell mass and a fluid-filled lumen, the blastocyst results from a series of cleavages divisions, morphogenetic movements and lineage specification. Recent studies identified the essential role of actomyosin contractility in driving the morphogenesis, fate specification and cytokinesis leading to the formation of the blastocyst. However, the preimplantation development of contractility mutants has not been characterized. Here, we generated single and double maternal-zygotic mutants of non-muscle myosin-II heavy chains (NMHC) to characterize them using multiscale imaging. We find that Myh9 (NMHC II-A) is the major NMHC during preimplantation development as its maternal-zygotic loss causes failed cytokinesis, increased duration of the cell cycle, weaker embryo compaction and reduced differentiation, whereas Myh10 (NMHC II-B) maternal-zygotic loss is much less severe. Double maternal-zygotic mutants for Myh9 and Myh10 show a much stronger phenotype, failing most attempts of cytokinesis. We find that morphogenesis and fate specification are affected but nevertheless carry on in a timely fashion, regardless of the impact of the mutations on cell number. Strikingly, even when all cell divisions fail, the resulting single-celled embryo can initiate trophectoderm differentiation and lumen formation by accumulating fluid in increasingly large vacuoles. Therefore, contractility mutants reveal that fluid accumulation is a cell-autonomous process and that the preimplantation program carries on independently of successful cell division.

181 MOUSE TRIPLETS DEVELOPED FROM SINGLE BLASTOMERES OF AN 8-CELL-STAGE EMBRYO SUPPORTED WITH TETRAPLOID EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 198
Author(s):  
C. V. Bogdan ◽  
A. P. Catunda ◽  
S. Bodo ◽  
D. Ilie ◽  
A. Kovacs ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Es Cells ◽  
Biological Effects ◽  
Cell Mass ◽  
Cell Stage ◽  
Stage Embryo ◽  
Tetraploid Embryo ◽  
Single Blastomere ◽  
Single Blastomeres ◽  
Diploid Cells

Chimera production using altered ES cells became a key tool for generating transgenic mice. However, chimeras are more than just a tool for making mouse mutants; they play a crucial role in analyzing the biological effects of genetic changes. Chimeras can be made by combining two whole 8-cell embryos or by combining subsets of blastomeres of two cleavage stage embryos. Because, at these stages, the early embryonic cells are not yet restricted in their lineage potency, they are equally capable of contributing to the inner cell mass or the trophectoderm. Pluripotency of single blastomeres of the 4-cell and 8-cell mouse embryo has been proved indirectly by aggregating them with carrier blastomers of a different genotype, giving rise to chimeric blastocyst (Tarkowski et al. 1967 J. Embryol. Exp. Morphol. 18, 155-180). In our study we wanted to demonstrate that a single blastomere of an 8-cell stage embryo, supported with tetraploid embryos at the 4-cell stage, is capable of developing into a healthy animal. When tetraploid embryos are used to make chimeras together with diploid cells, tetraploid cells rarely contribute to the embryo itself, but contribute mainly to the primitive endoderm and the trophectoderm, so in this case the newborns will be derived only from the diploid blastomers used (Nagy et al. 1990 Development 110, 815-821). We produced chimeras by aggregating a single blastomere [(2n)(1-cell)] derived by combining sexed, GFP-expressing, diploid 8-cell stage embryo (Hadjantonakis et al. 1998 Nat. Genet. 19, 220-222) with either a sexed diploid 7-cell embryo [(2n)(7-cells)] (one of the 8-cells was taken for sexing) or a non-sexed tetraploid embryo [(4n)(4-cells)]. The aggregates were cultured in vitro and transferred as blastocysts to the uteruses of pseudo-pregnant females. Fetuses were removed by Caesarian section and raised by lactating foster mothers. From the transferred 84 [XY(2n)(1-cell)]/[XX(2n)(7-cells)] aggregates we obtained 12 (14.3%) newborns, 11 (91.7%) males and one (8.3%) female. From the transferred 27 [XY(2n)(1-cell)]/[(4n)(4-cells)] aggregates, where one XY blastomere was combined with a tetraploid embryo, we obtained 7 (25.9%) male newborns: 1 triplet and 2 pairs of twins. We also transferred four [XX(2n)(1-cell)]/[(4n)(4-cells)] chimera embryos, where an XX-blastomere was aggregated with a tetraploid embryo; we obtained a set of living female triplets (75%) from this aggregate. We demonstrated that a single blastomere of the 8-cell-stage embryo is capable of developing into a living newborn. We obtained identical pups from three blastomeres isolated from the same embryo (triplets). This way we produced single blastomere clones and could control the sex of the new generation. This research was supported by grants from OTKA T037582 and RO-1/2002 Intergovernmental S&T cooperation program.

Generation of embryonic stem cells: limitations of and alternatives to inner cell mass harvest

2008 ◽  
Vol 24 (3-4) ◽  
pp. E4 ◽  
Author(s):  
Sunit Das ◽  
Michael Bonaguidi ◽  
Kenji Muro ◽  
John A. Kessler
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Ethical Considerations ◽  
Pluripotent Cells ◽  
Mammalian Embryo ◽  
Induced Pluripotency ◽  
Early Mammalian Embryo

✓ Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the early mammalian embryo. Because of their plasticity and potentially unlimited capacity for self-renewal, ES cells have generated tremendous interest both as models for developmental biology and as possible tools for regenerative medicine. This excitement has been attenuated, however, by scientific, political, and ethical considerations. In this article the authors describe somatic cell nuclear transfer and transcription-induced pluripotency, 2 techniques that have been used in attempts to circumvent the need to derive ES cells by the harvest of embryonic tissue.

scholarly journals A multiscale model via single-cell transcriptomics reveals robust patterning mechanisms during early mammalian embryo development

2021 ◽  
Vol 17 (3) ◽  
pp. e1008571
Author(s):  
Zixuan Cang ◽  
Yangyang Wang ◽  
Qixuan Wang ◽  
Ken W. Y. Cho ◽  
William Holmes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Mammalian Embryo ◽  
Early Mammalian Embryo ◽  
Cell Gene Expression ◽  
Cell Gene ◽  
Cell Cell

During early mammalian embryo development, a small number of cells make robust fate decisions at particular spatial locations in a tight time window to form inner cell mass (ICM), and later epiblast (Epi) and primitive endoderm (PE). While recent single-cell transcriptomics data allows scrutinization of heterogeneity of individual cells, consistent spatial and temporal mechanisms the early embryo utilize to robustly form the Epi/PE layers from ICM remain elusive. Here we build a multiscale three-dimensional model for mammalian embryo to recapitulate the observed patterning process from zygote to late blastocyst. By integrating the spatiotemporal information reconstructed from multiple single-cell transcriptomic datasets, the data-informed modeling analysis suggests two major processes critical to the formation of Epi/PE layers: a selective cell-cell adhesion mechanism (via EphA4/EphrinB2) for fate-location coordination and a temporal attenuation mechanism of cell signaling (via Fgf). Spatial imaging data and distinct subsets of single-cell gene expression data are then used to validate the predictions. Together, our study provides a multiscale framework that incorporates single-cell gene expression datasets to analyze gene regulations, cell-cell communications, and physical interactions among cells in complex geometries at single-cell resolution, with direct application to late-stage development of embryogenesis.

scholarly journals MED20 is essential for early embryogenesis and regulates NANOG expression

Reproduction ◽  
2019 ◽  
Vol 157 (3) ◽  
pp. 215-222 ◽  
Author(s):  
Wei Cui ◽  
Chelsea Marcho ◽  
Yongsheng Wang ◽  
Rinat Degani ◽  
Morgane Golan ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Blastocyst Stage ◽  
Mediator Complex ◽  
Mammalian Development ◽  
Lineage Specification ◽  
Mammalian Embryo ◽  
Pol Ii

Mediator is an evolutionarily conserved multi-subunit complex, bridging transcriptional activators and repressors to the general RNA polymerase II (Pol II) initiation machinery. Though the Mediator complex is crucial for the transcription of almost all Pol II promoters in eukaryotic organisms, the phenotypes of individual Mediator subunit mutants are each distinct. Here, we report for the first time, the essential role of subunit MED20 in early mammalian embryo development. Although Med20 mutant mouse embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at early post-gastrulation stages. Outgrowth assays show that mutant blastocysts cannot hatch from the zona pellucida, indicating impaired blastocyst function. Assessments of cell death and cell lineage specification reveal that apoptosis, inner cell mass, trophectoderm and primitive endoderm markers are normal in mutant blastocysts. However, the epiblast marker NANOG is ectopically expressed in the trophectoderm of Med20 mutants, indicative of defects in trophoblast specification. These results suggest that MED20 specifically, and the Mediator complex in general, are essential for the earliest steps of mammalian development and cell lineage specification.

scholarly journals Multiscale analysis of single and double maternal-zygotic Myh9 and Myh10 mutants during mouse preimplantation development

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Markus Frederik Schliffka ◽  
Anna-Francesca Tortorelli ◽  
Özge Özgüç ◽  
Ludmilla de Plater ◽  
Oliver Polzer ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Preimplantation Development ◽  
Mammalian Development ◽  
Mammalian Embryo ◽  
Fate Specification ◽  
Heavy Chains ◽  
A Cell ◽  
The Impact

During the first days of mammalian development, the embryo forms the blastocyst, the structure responsible for implanting the mammalian embryo. Consisting of an epithelium enveloping the pluripotent inner cell mass and a fluid-filled lumen, the blastocyst results from a series of cleavages divisions, morphogenetic movements and lineage specification. Recent studies identified the essential role of actomyosin contractility in driving the cytokinesis, morphogenesis and fate specification leading to the formation of the blastocyst. However, the preimplantation development of contractility mutants has not been characterized. Here, we generated single and double maternal-zygotic mutants of non-muscle myosin II heavy chains (NMHC) to characterize them with multiscale imaging. We find that Myh9 (NMHC II-A) is the major NMHC during preimplantation development as its maternal-zygotic loss causes failed cytokinesis, increased duration of the cell cycle, weaker embryo compaction and reduced differentiation, whereas Myh10 (NMHC II-B) maternal-zygotic loss is much less severe. Double maternal-zygotic mutants for Myh9 and Myh10 show a much stronger phenotype, failing most attempts of cytokinesis. We find that morphogenesis and fate specification are affected but nevertheless carry on in a timely fashion, regardless of the impact of the mutations on cell number. Strikingly, even when all cell divisions fail, the resulting single-celled embryo can initiate trophectoderm differentiation and lumen formation by accumulating fluid in increasingly large vacuoles. Therefore, contractility mutants reveal that fluid accumulation is a cell-autonomous process and that the preimplantation program carries on independently of successful cell division.

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