Transcriptome analyses of inner cell mass and trophectoderm cells isolated by magnetic-activated cell sorting from bovine blastocysts using single cell RNA-seq

2016 ◽  
Vol 51 (5) ◽  
pp. 726-735 ◽  
Author(s):  
X-M Zhao ◽  
L-S Cui ◽  
H-S Hao ◽  
H-Y Wang ◽  
S-J Zhao ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Sorting ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Rna Seq ◽  
Trophectoderm Cells ◽  
Transcriptome Analyses ◽  
Magnetic Activated Cell Sorting

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 Tracing the Derivation of Embryonic Stem Cells from the Inner Cell Mass by Single-Cell RNA-Seq Analysis

2010 ◽  
Vol 6 (5) ◽  
pp. 468-478 ◽  
Author(s):  
Fuchou Tang ◽  
Catalin Barbacioru ◽  
Siqin Bao ◽  
Caroline Lee ◽  
Ellen Nordman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Rna Seq

Interactions of blastomeres suggest changes in cell surface adhesiveness during the formation of inner cell mass and trophectoderm in the preimplantation mouse embryo

Development ◽  
1982 ◽  
Vol 70 (1) ◽  
pp. 133-152
Author(s):  
Susan J. Kimber ◽  
M. Azim ◽  
H. Surani ◽  
Sheila C. Barton
Keyword(s):  
Inner Cell Mass ◽  
Single Cells ◽  
Cell Mass ◽  
Cell Stage ◽  
Trophectoderm Cells ◽  
Adhesive Surface ◽  
Preimplantation Mouse Embryo ◽  
Divergent Differentiation ◽  
Preimplantation Mouse ◽  
The Difference

Whole 8-cell morulae can be aggregated with isolated inner cell masses from blastocysts. On examining semithin light microscope sections of such aggregates we found that cells of the morula changed shape and spread over the surface of the ICM, thus translocating it to the inside of the aggregate. Using single cells from 8-cell embryos in combination with single cells from other stage embryos or isolated ICMs we show that 1/8 blastomeres spread over other cells providing a suitably adhesive surface. The incidence of spreading is high with inner cells from 16-cell embryos (56 %) and 32-cell embryos (62%) and isolated inner cell masses (64%). In contrast, the incidence of spreading of 1/8 blastomeres is low over outer cells from 16-cell embryos (26%) and 32-cell embryos (13%). Blastomeres from 8-cell embryos do not spread over unfertilized 1-cell eggs, 1/2 or 1/4 cells or trophectoderm cells contaminating isolated ICMs. When 1/8 cells are aggregated in pairs they flatten on one another (equal spreading) as occurs at compaction in whole 8-cell embryos. However, if 1/8 is allowed to divide to 2/16 in culture one of the cells engulfs the other (51-62/ pairs). Based on the ideas of Holtfreter (1943) and Steinberg (1964,1978) these results are interpreted to indicate an increase in adhesiveness at the 8-cell stage as well as cytoskeletal mobilization. Following the 8-cell stage there is an increase in adhesiveness of inside cells while the outside cells decrease in adhesiveness. The difference in adhesiveness between inside and outside cells in late morulae is probably central to the divergent differentiation of (inner) ICM and (outer) trophectoderm cell populations.

scholarly journals Base editing in bovine embryos reveals a species-specific role of SOX2 in regulation of pluripotency

2021 ◽  
Author(s):  
Lei Luo ◽  
Yan Shi ◽  
Huanan Wang ◽  
Zizengchen Wang ◽  
Yanna Dang ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Rna Seq ◽  
Bovine Embryos ◽  
Base Editing ◽  
Sox2 Expression ◽  
Nanog Expression ◽  
Species Specific ◽  
Lineage Specific Genes

The emergence of the first three lineages during development are orchestrated by a network of transcription factors, which are best characterized in mice. However, the role and regulation of these factors are not completely conserved in other mammals, including human and cattle. Here, we establish a gene inactivation system by introducing premature codon with cytosine base editor in bovine embryos with a robust efficiency. Of interest, SOX2 is universally localized in early blastocysts but gradually restricted into the inner cell mass in cattle. SOX2 knockout results in a failure of the establishment of pluripotency. Indeed, OCT4 level is significantly reduced and NANOG was barely detectable. Furthermore, the formation of primitive endoderm is compromised with few SOX17 positive cells. Single embryo RNA-seq reveals a dysregulation of 2074 genes, among which 90% are up-regulated in SOX2-null blastocysts. Intriguingly, more than a dozen lineage-specific genes, including OCT4 and NANOG, are down-regulated. Moreover, SOX2 expression is sustained in the trophectoderm in absence of CDX2 in bovine late blastocysts. Overall, we propose that SOX2 is dispensable for OCT4 and NANOG expression and disappearance of SOX2 in the trophectoderm depends on CDX2 in cattle, which are all in sharp contrast with results in mice.

Trophectodermal processes regulate the expression of totipotency within the inner cell mass of the mouse expanding blastocyst

Development ◽  
1984 ◽  
Vol 84 (1) ◽  
pp. 63-90
Author(s):  
Tom P. Fleming ◽  
Paul D. Warren ◽  
Julia C. Chisholm ◽  
Martin H. Johnson
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Central Area ◽  
Ultrastructural Changes ◽  
Trophectoderm Cells ◽  
Junctional Complexes ◽  
Secondary Lysosomes ◽  
Cytoplasmic Processes ◽  
Cellular Cover

Mouse blastocysts, aged 0, 2, 6 and 12 h from the onset of cavitation, were examined by transmission (TEM) and scanning (SEM) electron microscopy. In TEM sections, trophectoderm cells (TE) differed morphologically from those of the inner cell mass (ICM) by their flattened shape, paler cytosol staining and polarized disposition of both junctional complexes (apicolateral) and intracellular secondary lysosomes (SL; basal). Throughout this period of development, cytoplasmic processes, characterized by abundant SLs, cover approximately 80 % of the juxtacoelic face of the ICM. These processes are shown to be derived from the basal surface of TE cells intermediately placed between polar and mural regions. In SEM preparations of the juxtacoelic ICM surface, revealed by ‘cracking open’ blastocysts, the processes appear as tongue-shaped, centripetally oriented structures which terminate collectively at a central area on the ICM surface. The potential of cultured ICMs to generate TE was demonstrated following their immunosurgical isolation from blastocysts aged up to 12 h post cavitation and by examining the sequence of ultrastructural changes associated with TE generation by ICMs from 2 h blastocysts. In contrast, the juxtacoelic cells of similarly aged ICMs observed in situ in ultrasections of intact embryos showed little or no evidence of totipotency expression as judged by the absence of TE characteristics. Since TE expression within presumptive ICM cells is thought to be generated by an asymmetry of cell contacts (Johnson & Ziomek, 1983), we propose that the juxtacoelic TE processes, by providing a cellular cover to the ICM, function in suppressing the expression in situ of ICM totipotency.

scholarly journals Lipid Stores and Lipid Metabolism Associated Gene Expression in Porcine and Bovine Parthenogenetic Embryos Revealed by Fluorescent Staining and RNA-seq

2020 ◽  
Vol 21 (18) ◽  
pp. 6488
Author(s):  
Arkadiusz Kajdasz ◽  
Ewelina Warzych ◽  
Natalia Derebecka ◽  
Zofia E. Madeja ◽  
Dorota Lechniak ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lipid Metabolism ◽  
Inner Cell Mass ◽  
Mammalian Species ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Rna Seq ◽  
Expression Of Genes ◽  
Bovine Blastocyst

Compared to other mammalian species, porcine oocytes and embryos are characterized by large amounts of lipids stored mainly in the form of droplets in the cytoplasm. The amount and the morphology of lipid droplets (LD) change throughout the preimplantation development, however, relatively little is known about expression of genes involved in lipid metabolism of early embryos. We compared porcine and bovine blastocyst stage embryos as well as dissected inner cell mass (ICM) and trophoblast (TE) cell populations with regard to lipid droplet storage and expression of genes functionally annotated to selected lipid gene ontology terms using RNA-seq. Comparing the number and the volume occupied by LD between bovine and porcine blastocysts, we have found significant differences both at the level of single embryo and a single blastomere. Aside from different lipid content, we found that embryos regulate the lipid metabolism differentially at the gene expression level. Out of 125 genes, we found 73 to be differentially expressed between entire porcine and bovine blastocyst, and 36 and 51 to be divergent between ICM and TE cell lines. We noticed significant involvement of cholesterol and ganglioside metabolism in preimplantation embryos, as well as a possible shift towards glucose, rather than pyruvate dependence in bovine embryos. A number of genes like DGAT1, CD36 or NR1H3 may serve as lipid associated markers indicating distinct regulatory mechanisms, while upregulated PLIN2, APOA1, SOAT1 indicate significant function during blastocyst formation and cell differentiation in both models.

Defects in the allocation of cells to the inner cell mass and trophectoderm of parthenogenetic mouse blastocysts

1996 ◽  
Vol 8 (8) ◽  
pp. 1193 ◽  
Author(s):  
B Mognetti ◽  
D Sakkas
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Substantial Reduction ◽  
Cell Lineage ◽  
Blastocyst Stage ◽  
Trophectoderm Cells ◽  
Parthenogenetic Development ◽  
Preimplantation Stage ◽  
Parthenogenetic Mouse Embryos ◽  
Parthenogenetic Embryos

Diploid parthenogenetic mouse embryos (which possess two maternally-derived genomes) can develop only as far as the 25-somite stage when transferred in utero and exhibit a substantial reduction in trophoblast tissue. The loss of cultured parthenogenetic embryos during postimplantation indicates that a defect in cell lineage may be evident as early as the blastocyst stage. The possibility that a defect may already be reflected at the preimplantation stage was investigated by examining the allocation of cells to the trophectoderm (trophoblast progenitor cells) and the inner cell mass of haploid and diploid parthenogenetic mouse blastocysts. Utilizing a differential labelling technique for counting cells, diploid parthenogenetic blastocysts were found to have fewer inner cell mass cells and trophectoderm cells than their haploid counterparts and normal blastocysts. In addition, both haploid and diploid parthenogenetic blastocysts had a lower inner cell mass: trophectoderm ratio than normal blastocysts. Thus, the relatively poor development of the trophectoderm lineage at the postimplantation stage is not reflected by a reduction in its allotment of cells at its first appearance. Nevertheless, the findings indicate that parthenogenetic development is already compromised at the blastocyst stage, and provide evidence that the expression of imprinted genes has significance for the development of the embryo at the preimplantation stage.

scholarly journals Oct4 regulates embryonic pluripotency via metabolic mechanisms and Stat3 signalling

2020 ◽  
Author(s):  
Giuliano Giuseppe Stirparo ◽  
Agata Kurowski ◽  
Stanley Eugene Strawbridge ◽  
Hannah Stuart ◽  
Thorsten Edwin Boroviak ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Specific Gene ◽  
List Type ◽  
Early Mouse

AbstractOCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 protects the pluripotent lineage from differentiation into trophoblast, we used single cell transcriptomics and quantitative immunofluorescence on blastocysts and established differentially expressed genes and pathways between control and OCT4 null cells. Activation of most pluripotency-associated transcription factors in the early mouse inner cell mass appears independent of OCT4, whereas JAK/STAT signalling requires OCT4, via activation of IL6ST. Single cell deconvolution, diffusion component and trajectory inference dissected the process of differentiation of OCT4 null cells by activating specific gene-network and transcription factors. Downregulation of glycolytic and oxidative metabolism was observed. CHIPseq analysis suggests OCT4 directly targets rate-limiting glycolytic enzymes. Concomitant with significant disruption of the STAT3 pathway, oxidative respiration is significantly diminished in OCT4 null cells. Upregulation of the lysosomal pathway detected in OCT4 null embryos is likely attributable to aberrant metabolism.Highlights and noveltyMajor pluripotency-associated transcription factors are activated in OCT4-deficient early mouse ICM cells, coincident with ectopic expression of trophectoderm markersJAK/STAT signalling is defective in OCT4 null embryosOCT4 promotes expression of KATS enzymes by means of glycolytic production of Acetyl CoA to secure chromatin accessibility for acquisition of epiblast identityOCT4 regulates the metabolic and biophysical processes required for establishment of embryonic pluripotency

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