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

scholarly journals Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 231-242 ◽  
Author(s):  
Craig Smith ◽  
Debbie Berg ◽  
Sue Beaumont ◽  
Neil T Standley ◽  
David N Wells ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Cell Number ◽  
Transcript Levels ◽  
Multiple Genes

During somatic cell nuclear transfer (NT), the transcriptional status of the donor cell has to be reprogrammed to reflect that of an embryo. We analysed the accuracy of this process by comparing transcript levels of four developmentally important genes (Oct4,Otx2,Ifitm3,GATA6), a gene involved in epigenetic regulation (Dnmt3a) and three housekeeping genes (β-actin, β-tubulinandGAPDH) in 21 NT blastocysts with that in genetically half-identicalin vitroproduced (IVP,n=19) andin vivo(n=15) bovine embryos. We have optimised an RNA-isolation and SYBR-green-based real-time RT-PCR procedure allowing the reproducible absolute quantification of multiple genes from a single blastocyst. Our data indicated that transcript levels did not differ significantly between stage and grade-matched zona-free NT and IVP embryos except for Ifitm3/Fragilis, which was expressed at twofold higher levels in NT blastocysts.Ifitm3expression is confined to the inner cell mass at day 7 blastocysts and to the epiblast in day 14 embryos. No ectopic expression in the trophectoderm was seen in NT embryos. Gene expression in NTand IVP embryos increased between two- and threefold for all eight genes from early to late blastocyst stages. This increase exceeded the increase in cell number over this time period indicating an increase in transcript number per cell. Embryo quality (morphological grading) was correlated to cell number for NT and IVP embryos with grade 3 blastocysts containing 30% fewer cells. However, only NT embryos displayed a significant reduction in gene expression (50%) with loss of quality. Variability in gene expression levels was not significantly different in NT, IVP orin vivoembryos but differed among genes, suggesting that the stringency of regulation is intrinsic to a gene and not affected by culture or nuclear transfer.Oct4levels exhibited the lowest variability. Analysing the total variability of all eight genes for individual embryos revealed thatin vivoembryos resembled each other much more than did NT and IVP blastocysts. Furthermore,in vivoembryos, consisting of 1.5-fold more cells, generally contained two- to fourfold more transcripts for the eight genes than did their cultured counterparts. Thus, culture conditions (in vivoversusin vitro) have greater effects on gene expression than does nuclear transfer when minimising genetic heterogeneity.

scholarly journals MCRS1 is essential for epiblast development during early mouse embryogenesis

Reproduction ◽  
2020 ◽  
Vol 159 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Wei Cui ◽  
Agnes Cheong ◽  
Yongsheng Wang ◽  
Yuran Tsuchida ◽  
Yong Liu ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Histone H4 ◽  
Histone H4 Acetylation ◽  
Early Mouse

Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.

305 CONSTRUCTION OF TRANSCRIPTION FACTOR GENES FOR REPROGRAMMING OF ADULT GOAT FIBROBLAST CELLS FOR PRODUCTION OF INDUCED PLURIPOTENT STEM CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 249
Author(s):  
D. Kumar ◽  
D. Malakar ◽  
R. Dutta ◽  
S. Garg ◽  
S. Sahu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Somatic Cells ◽  
Ectopic Expression ◽  
Fibroblast Cells ◽  
Induced Pluripotent

Embryonic stem cells (ESC) are derived from the inner cell mass of blastocysts and proliferate extensively while maintaining pluripotency. They can be used for the treatment of juvenile diabetes, Parkinson’s disease, heart failure, and spinal cord injury. However, the use of embryos and tissue rejection remain concerns for ESC transplantation. Reprogramming of somatic cells may be done by different methods such as somatic cell nuclear transfer (Wilmut et al. 1997), fusion of somatic cells (Cowen et al. 2005), treatment with the extract of the pluripotent stem cells (Johnson Rajasingh 2008), and by the stable ectopic expression of defined factors in the somatic cells (Takahashi and Yamanaka 2006). Several transcription factors, including Oct3/4 (Nichols et al. 1998; Niwa et al. 2000), Sox2 (Avilion et al. 2003), and Nanog (Chambers et al. 2003; Mitsui et al. 2003), function in the maintenance of pluripotency in both early embryos and ESC. Takahashi and Yamanaka reported reprogramming the fibroblast cells into stem cells by introducing Oct3/4, Sox2, c-Myc, and Klf4 in mouse embryonic and adult fibroblasts. Yu et al. (2007) demonstrated that four transcription factors (OCT-4, SOX2, NANOG, and LIN28) are sufficient to reprogramme human somatic cells to pluripotent stem cells that exhibit the essential characteristics of ESC. Nakagawa et al. (2008) used three factors (OCT3/4, SOX2, and KLF4) for human iPS cell production from somatic cells. We are trying to reprogramme the adult goat fibroblast cells in induced pluripotent stem cells by using ectopic expression of transcription factors such as Oct-4, Sox2, Nanog, and Lin28. We collected the ovaries from a slaughtered animal from Delhi and collected the oocytes from ovaries. Then after the collection, A and B grade oocytes were selected. Selected oocytes were processed and incubated in in vitro maturation media for 24 h. We collected semen from a male goat, and it was processed and capacitated in sperm TALP. Capacitated sperms were used for IVF of the in vitro matured oocytes in ferTALP. After 12 h sperm were washed from oocytes in embryo developing media (EDM), and oocytes were cultured (in vitro) in EDM. After 24 h cleavage occurred. The cleaved embryos were cultured for 6 to 7 days. At the 7th day, we got blastocysts. From these blastocysts, inner cell mass was isolated enzymatically and cultured to get ESC. The ESC were cultured for 7 passages and used for RNA isolation. The RNA was isolated from these stem cells by the Trizol method. Complementary DNA was prepared by RT-PCR. Using gene-specific primer for Oct-4, Nanog, and Sox2, DNA was amplified. The DNA for the Oct-4, Nanog, and Sox2 genes was cloned in pJET cloning vector and transformed in Top10 E. coli competence cells. After screening, plasmid was isolated and sent for sequencing. Sequences were analysed and the complete open reading frame was created for Oct-4, Nanog, and Sox2.

scholarly journals Loss of RBBP4 results in defective inner cell mass, severe apoptosis, hyperacetylated histones and preimplantation lethality in mice†

2020 ◽  
Vol 103 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Xiaosu Miao ◽  
Tieqi Sun ◽  
Holly Barletta ◽  
Jesse Mager ◽  
Wei Cui
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Lineage Specification ◽  
Mouse Embryo Development ◽  
Evolutionarily Conserved ◽  
Early Mouse

Abstract Retinoblastoma-binding protein 4 (RBBP4) (also known as chromatin-remodeling factor RBAP48) is an evolutionarily conserved protein that has been involved in various biological processes. Although a variety of functions have been attributed to RBBP4 in vitro, mammalian RBBP4 has not been studied in vivo. Here we report that RBBP4 is essential during early mouse embryo development. Although Rbbp4 mutant embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at E7.5 early post-gastrulation stage, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts cannot hatch from the zona or can hatch but then arrest without further development. We find that while there is no change in proliferation or levels of reactive oxygen species, both apoptosis and histone acetylation are significantly increased in mutant blastocysts. Analysis of lineage specification reveals that while the trophoblast is properly specified, both epiblast and primitive endoderm lineages are compromised with severe reductions in cell number and/or specification. In summary, these findings demonstrate the essential role of RBBP4 during early mammalian embryogenesis.

Ratio and number of inner cell mass and trophoblast cells of in vitro and in vivo produced porcine embryos

1995 ◽  
Vol 43 (1) ◽  
pp. 304 ◽  
Author(s):  
D. Rath ◽  
H. Niemann ◽  
T. Tao ◽  
M. Boerjan
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Trophoblast Cells

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 793-803 ◽  
Author(s):  
V.E. Papaioannou ◽  
K.M. Ebert
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Staining Technique ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Morphological Development ◽  
Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

The control of trophoblastic growth in the guinea pig

Development ◽  
1980 ◽  
Vol 60 (1) ◽  
pp. 405-418
Author(s):  
E. B. Ilgren
Keyword(s):  
Guinea Pig ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
The Other ◽  
In Vivo Analysis

The growth of mouse trophectoderm depends upon the presence of the inner cell mass. Whether this applies to other species of mammals is not known. To investigate this problem, the guinea pig was selected for two reasons. Firstly, the growth of guinea-pig trophoblast resembles that of man. Secondly, earlier studies suggest that the proliferation of guinea-pig trophectoderm may not be under ICM control. Therefore, in the present study, the guinea-pig blastocyst was cut microsurgically to yield two tissue fragments. These contained roughly equal numbers of trophectodermal cells, one fragment being composed only of trophectoderm and the other containing ICM tissue as well. Subsequently, the growth of these mural and polar fragments was followed in vitro since numerous technical difficulties make an in vivo analysis of this problem impracticable. In a manner similar to the mouse, the isolated mural trophectoderm of the guinea pig stopped dividing and became giant. In contrast, guinea-pig polar fragments formed egg-cylinder-like structures. The latter contained regions structurally similar to two presumptive polar trophectodermal derivatives namely the ectoplacental and extraembryonic ectodermal tissues. These findings suggest that guinea-pig trophectodermal growth may occur in a manner similar to the mouse and thus be under ICM control.

scholarly journals Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3111
Author(s):  
Po-Yu Lin ◽  
Denny Yang ◽  
Chi-Hsuan Chuang ◽  
Hsuan Lin ◽  
Wei-Ju Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Developmental Potential ◽  
Functional Features ◽  
Early Mouse ◽  
Extraembryonic Tissues

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are ‘true’ totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely ‘cluster 3’, as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells.

Derivation of human embryonic stem cell lines after blastocyst microsurgery

2010 ◽  
Vol 88 (3) ◽  
pp. 479-490 ◽  
Author(s):  
Guoliang Meng ◽  
Shiying Liu ◽  
Xiangyun Li ◽  
Roman Krawetz ◽  
Derrick E. Rancourt
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Clinical Medicine ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Hesc Lines

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. Because of their ability to differentiate into a variety of cell types, human embryonic stem cells (hESCs) provide an unlimited source of cells for clinical medicine and have begun to be used in clinical trials. Presently, although several hundred hESC lines are available in the word, only few have been widely used in basic and applied research. More and more hESC lines with differing genetic backgrounds are required for establishing a bank of hESCs. Here, we report the first Canadian hESC lines to be generated from cryopreserved embryos and we discuss how we navigated through the Canadian regulatory process. The cryopreserved human zygotes used in this study were cultured to the blastocyst stage, and used to isolate ICM via microsurgery. Unlike previous microsurgery methods, which use specialized glass or steel needles, our method conveniently uses syringe needles for the isolation of ICM and subsequent hESC lines. ICM were cultured on MEF feeders in medium containing FBS or serum replacer (SR). Resulting outgrowths were isolated, cut into several cell clumps, and transferred onto fresh feeders. After more than 30 passages, the two hESC lines established using this method exhibited normal morphology, karyotype, and growth rate. Moreover, they stained positively for a variety of pluripotency markers and could be differentiated both in vitro and in vivo. Both cell lines could be maintained under a variety of culture conditions, including xeno-free conditions we have previously described. We suggest that this microsurgical approach may be conducive to deriving xeno-free hESC lines when outgrown on xeno-free human foreskin fibroblast feeders.

scholarly journals Early Embryonic Lethality of Mice Lacking the Essential Protein SNEV

2007 ◽  
Vol 27 (8) ◽  
pp. 3123-3130 ◽  
Author(s):  
Klaus Fortschegger ◽  
Bettina Wagner ◽  
Regina Voglauer ◽  
Hermann Katinger ◽  
Maria Sibilia ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Replicative Senescence ◽  
Inner Cell Mass ◽  
Umbilical Vein ◽  
Cell Mass ◽  
Preimplantation Embryos ◽  
Proliferative Potential ◽  
Mouse Development

ABSTRACT SNEV (Prp19, Pso4, NMP200) is a nuclear matrix protein known to be involved in pre-mRNA splicing, ubiquitylation, and DNA repair. In human umbilical vein endothelial cells, SNEV overexpression delayed the onset of replicative senescence. Here we analyzed the function of the mouse SNEV gene in vivo by employing homologous recombination in mice and conclude that SNEV is indispensable for early mouse development. Mutant preimplantation embryos initiated blastocyst formation but died shortly thereafter. Outgrowth of SNEV-null blastocysts showed a lack of proliferation of cells of the inner cell mass, which subsequently underwent cell death. While SNEV-heterozygous mice showed no overt phenotype, heterozygous mouse embryonic fibroblast cell lines with reduced SNEV levels displayed a decreased proliferative potential in vitro. Our experiments demonstrate that the SNEV protein is essential, functionally nonredundant, and indispensable for mouse development.

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