scholarly journals Imaging proprotein convertase activities and their regulation in the implanting mouse blastocyst

2010 ◽  
Vol 191 (1) ◽  
pp. 129-139 ◽  
Author(s):  
Daniel Mesnard ◽  
Daniel B. Constam
Keyword(s):  
Cell Surface ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Axis Formation ◽  
Fluorescent Biosensor ◽  
Membrane Bound ◽  
Biosensor Cell

Axis formation and allocation of pluripotent progenitor cells to the germ layers are governed by the TGF-β–related Nodal precursor and its secreted proprotein convertases (PCs) Furin and Pace4. However, when and where Furin and Pace4 first become active have not been determined. To study the distribution of PCs, we developed a novel cell surface–targeted fluorescent biosensor (cell surface–linked indicator of proteolysis [CLIP]). Live imaging of CLIP in wild-type and Furin- and Pace4-deficient embryonic stem cells and embryos revealed that Furin and Pace4 are already active at the blastocyst stage in the inner cell mass and can cleave membrane-bound substrate both cell autonomously and nonautonomously. CLIP was also cleaved in the epiblast of implanted embryos, in part by a novel activity in the uterus that is independent of zygotic Furin and Pace4, suggesting a role for maternal PCs during embryonic development. The unprecedented sensitivity and spatial resolution of CLIP opens exciting new possibilities to elucidate PC functions in vivo.

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.

145. TRP53 REGULATES THE FORMATION OF A PLURIPOTENT INNER CELL MASS IN THE EARLY EMBRYO

2009 ◽  
Vol 21 (9) ◽  
pp. 63
Author(s):  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Pluripotent Cells ◽  
Drug Induced ◽  
Developmental Potential

The developmental viability of the early embryo requires the formation of the inner cell mass (ICM) at the blastocyst stage. The ICM contributes to all cell lineages within the developing embryo in vivo and the embryonic stem cell (ESC) lineage in vitro. Commitment of cells to the ICM lineage and its pluripotency requires the expression of core transcription factors, including Nanog and Pou5f1 (Oct4). Embryos subjected to culture in vitro commonly display a reduced developmental potential. Much of this loss of viability is due to the up-regulation of TRP53 in affected embryos. This study investigated whether increased TRP53 disrupts the expression of the pluripotency proteins and the normal formation of the ICM lineage. Mouse C57BL6 morulae and blastocysts cultured from zygotes (modHTF media) possessed fewer (p < 0.001) NANOG-positive cells than equivalent stage embryos collected fresh from the uterus. Blocking TRP53 actions by either genetic deletion (Trp53–/–) or pharmacological inhibition (Pifithrin-α) reversed this loss of NANOG expression during culture. Zygote culture also resulted in a TRP53-dependent loss of POU5F1-positive cells from resulting blastocysts. Drug-induced expression of TRP53 (by Nutlin-3) also caused a reduction in formation of pluripotent ICM. The loss of NANOG- and POU5F1-positive cells caused a marked reduction in the capacity of blastocysts to form proliferating ICM after outgrowth, and a consequent reduced ability to form ESC lines. These poor outcomes were ameliorated by the absence of TRP53, resulting in transmission distortion in favour of Trp53–/– zygotes (p < 0.001). This study shows that stresses induced by culture caused TRP53-dependent loss of pluripotent cells from the early embryo. This is a cause of the relative loss of viability and developmental potential of cultured embryos. The preferential survival of Trp53–/– embryos after culture due to their improved formation of pluripotent cells creates a genetic danger associated with these technologies.

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.

166 EXPRESSION OF PLURIPOTENCY-DETERMINING FACTORS IN IN VITRO-PRODUCED BUFFALO EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 163
Author(s):  
T. Anand ◽  
D. Kumar ◽  
M. K. Singh ◽  
M. S. Chauhan ◽  
R. S. Manik ◽  
...  
Keyword(s):  
Cell Surface ◽  
Expression Patterns ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Tumor Rejection ◽  
Cell Surface Expression ◽  
Human Embryos ◽  
Strong Expression

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of blastocysts. These are pluripotent cells that retain the ability to differentiate into all cell types. Various cell surface antigens, the expressions of which have been widely used as markers to monitor the pluripotency of ESCs, include Oct-4, stage-specific embryonic antigens (SSEAs) such as SSEA-1, SSEA-3, and SSEA-4, and tumor rejection antigens (TRAs) such as TRA-1-60 and TRA-1-81. In this study, the cell surface expression patterns of these markers were examined in in vitro-produced buffalo embryos at the 2-, 4-, 8- to 16-cell, morula, and blastocyst stages using immunofluorescence microscopy. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF, following which the cleaved embryos were cultured for 9 days for production of embryos at different stages (n = 246). The embryos were fixed in 4% paraformaldehyde in Dulbecco's phosphate-buffered saline (DPBS) for 30 min, permeabilized by treatment with 0.1% Triton X-100 in DPBS for 30 min, and incubated first with the blocking solution (4% normal goat serum) for 30 min and then with the primary antibody (Oct-4: clone 9E3; SSEA-1: MC-480; SSEA-3: MC-631; SSEA-4: MC-813-70; TRA-1-60: clone TRA-1-60; and TRA-1-81: clone TRA-1-81, Chemicon� Inc., Temecula, CA, USA) at a dilution of 1:10 to 1:20 for 1 h. After being washed with DPBS, the embryos were incubated with appropriate FITC-labeled second antibody (anti-rat IgM or anti-mouse IgG or IgM, diluted 1:100 to 1:200) for 1 h and then examined under a fluorescence microscope. Oct-4 expression was detected at all embryonic stages starting from the 2-cell to the blastocyst stage, in which ICM, but not trophectoderm cells, exhibited a strong expression. SSEA-4 signal was found to be strongest at the 2-cell stage, with continued expression at all intermediate stages until the blastocyst stage in which there was a strong expression in ICM cells. In contrast, all of the embryonic stages were found to be negative for SSEA-3 expression. The SSEA-1 signal was present at all of the embryonic stages but was very weak. Expression of TRA-1-60 and TRA-1-81, which was detected only on the inner surface of the zona pellucida and in the perivitelline space in early embryonic stages, was absent in morulae and blastocysts. The results of this study indicate that the pluripotency-determining markers are differentially expressed in buffalo embryos and that the pattern of their expression is distinct from that of murine and human embryos but resembles to some extent that of goat embryos. Comparison of the expression pattern of these markers needs to be done between embryonic cells and ESCs for a better understanding of their developmental regulation.

185 THE PORCINE EPIBLAST AND NOT THE INNER CELL MASS HAS DEVELOPED CONVENTIONAL PATHWAYS FOR REGULATION OF PLURIPOTENCY

2009 ◽  
Vol 21 (1) ◽  
pp. 191
Author(s):  
V. J. Hall ◽  
J. Christensen ◽  
P. Maddox-Hyttel
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Positive Control ◽  
Total Rna ◽  
Weak Expression ◽  
Whole Transcriptome

Pluripotency in mice and human embryonic stem cells is regulated by a number of transcription factors, notably including Oct-4, Sox-2, and Nanog. However, in the pig, previous research indicates that Oct-4 protein and mRNA is not specifically localized to the inner cell mass (ICM) of the zona-intact (ZI) blastocyst. Levels of expression of Nanog mRNA, on the other hand, appear to be low in the ZI blastocyst, and protein has not been detected. Similarly, Sox-2 expression in the ZI blastocyst is relatively low and not specific to the ICM. In this study, we investigated the mRNA expression of Oct-4, Sox-2, and Nanog in D6/D7-derived ZI porcine in vivo-derived blastocysts compared with epiblasts mechanically isolated from hatched D10/D11 in vivo-derived blastocysts. We then investigated components involved in pathways important for regulating pluripotency, including JAK/STAT (i.e. gp130, LIFr), FGF (i.e. bFGF, FGFr1, FGFr2), and BMP (bmp4, smad4) signaling pathways and their downstream targets, stat3, c-myc, c-fos, by using RT-PCR. Sows were artificially inseminated, and embryos were flushed from uteri following slaughter. Single D6/D7 blastocysts (n = 3), single mechanically isolated D10/D11 epiblasts (n = 3), endometrium, and oviduct total RNA was isolated using the RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Total RNA from the blastocysts and epiblasts was then amplified to form cDNA using the QuantiTect Whole Transcriptome kit (Qiagen). Positive control tissues (oviduct and endometrium) were reverse transcribed using the RevertAid First Strand cDNA synthesis kit (Fermentas, Burlington, Ontario, Canada). Primers were designed to span introns in highly homologous sequences to human mRNA. Primers were tested in both oviduct and endometrium tissue, and products were sequenced to confirm specificity. PCR was performed at 55°C for 35 cycles. Results indicate that D6/D7 blastocysts only expressed Oct-4 and not Nanog and Sox-2. In contrast, all 3 transcripts were expressed in D10/D11 epiblasts. The D10/D11 epiblasts also expressed LIFr, bFGF, FGFr1, FGFr2, bmp4, smad4, stat3, c-myc, and c-fos. The cytokine receptor gp130 was only weakly expressed in a single epiblast. In contrast, the earlier stage D6/D7 blastocysts failed to express these messengers with the exception of weak expression of gp130 in all 3 blastocysts, and only a single blastocyst expressed LIFr, smad4, c-myc, and c-fos. In conclusion, this study indicates that the ICM of the porcine D6/D7 ZI blastocyst has not developed pluripotency signaling as observed in mice and humans at this developmental stage. Furthermore, without expression of gp130, the JAK/STAT pathway is unlikely to play a role in regulating pluripotency in the epiblast. It is likely that the later stage epiblast may be more amenable for the derivation of porcine embryonic stem cells.

In vivo and in vitro development of mouse embryos homozygous for the embryonic lethal velvet coat (Ve) mutation

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 43-55
Author(s):  
J. Rossant ◽  
K. M. Vijh
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Giant Cells ◽  
Blastocyst Stage ◽  
Parietal Endoderm ◽  
Trophoblast Giant Cells ◽  
Vitro Development ◽  
Ectoplacental Cone

Embryos homozygous for the velvet coat mutation, Ve/Ve, were recognized at 6·5 days post coitum by the reduced size of the ectodermal portions of the egg cylinder and the loose, columnar nature of the overlying endoderm. Later in development ectoderm tissues were sometimes entirely absent. Abnormalities appeared in the ectoplacental cone at 8·5 days but trophoblast giant cells and parietal endoderm appeared unaffected. Homozygotes could not be unequivocally identified at 5·5 days nor at the blastocyst stage but were recognized in blastocyst outgrowths by poor development of the inner cell mass derivatives, It has previously been suggested that Ve may exert its action at the blastocyst stage by reducing the size of the inner cell mass, but no evidence for such a reduction was found. Most of the observations on Ve/Ve homozygotes are, however, consistent with the hypothesis that Ve exerts its action primarily on later primitive ectoderm development.

Production and analysis of ES cell aggregation chimeras

1999 ◽  
Author(s):  
Andras Nagy ◽  
Janet Rossant
Keyword(s):  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Aggregation ◽  
Blastocyst Stage ◽  
Original Method ◽  
Embryonic Cells ◽  
Es Cell ◽  
Cleavage Stage

Embryonic stem (ES) cells behave like normal embryonic cells when returned to the embryonic environment after injection into a host blastocyst or after aggregation with earlier blastomere stage embryos. In such chimeras, ES cells behave like primitive ectoderm or epiblast cells (1), in that they contribute to all lineages of the resulting fetus itself, as well as to extraembryonic tissues derived from the gastrulating embryo, namely the yolk sac mesoderm, the amnion, and the allantois. However, even when aggregated with preblastocyst stage embryos, ES cells do not contribute to derivatives of the first two lineages to arise in development, namely, the extraembryonic lineages: trophoblast and primitive endoderm (2). The pluripotency of ES cells within the embryonic lineages is critical to their use in introducing new genetic alterations into mice, because truly pluripotent ES cells can contribute to the germline of chimeras, as well as all somatic lineages. However, the ability of ES cells to co-mingle with host embryonic cells, specifically in the embryonic, but not the major extraembryonic lineages, opens up a variety of possibilities for analysing gene function by genetic mosaics rather than by germline mutant analysis alone (3). There are two basic methods for generating pre-implantation chimeras in mice, whether it be embryo ↔ embryo or ES cell ↔ embryo chimeras. Blastocyst injection, in which cells are introduced into the blastocoele cavity using microinjection pipettes and micromanipulators, has been the method of choice for most ES cell chimera work (see Chapter 4). However, the original method for generating chimeras in mice, embryo aggregation, is considerably simpler and cheaper to establish in the laboratory. Aggregation chimeras are made by aggregating cleavage stage embryos together, or inner cell mass (ICM) or ES cells with cleavage stage embryos, growing them in culture to the blastocyst stage, and then transferring them to the uterus of pseudopregnant recipients to complete development. This procedure can be performed very rapidly by hand under the dissecting microscope, thus making possible high throughput production with minimal technical skill (4). In this chapter we describe some of the uses of pre-implantation chimeras, whether made by aggregation or blastocyst injection, but focus on the technical aspects of aggregation chimera generation. We also discuss the advantages and disadvantages of aggregation versus blastocyst injection for chimera production.

scholarly journals Development of Preimplantation Mouse Embryos in vivo and in vitro

1982 ◽  
Vol 35 (2) ◽  
pp. 187 ◽  
Author(s):  
GM Harlow ◽  
P Quinn
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

scholarly journals Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Sergey Rodin ◽  
Liselotte Antonsson ◽  
Colin Niaudet ◽  
Oscar E. Simonson ◽  
Elina Salmela ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Types ◽  
A Cell ◽  
Hes Cells ◽  
Free Environment ◽  
Different Cell Types ◽  
E Cadherin

Abstract Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

scholarly journals Derivation of Porcine Extra-Embryonic Endoderm Cell Lines Reveals Distinct Signaling Pathway and Multipotency States

2021 ◽  
Vol 22 (23) ◽  
pp. 12918
Author(s):  
Man-Ling Zhang ◽  
Yong Jin ◽  
Li-Hua Zhao ◽  
Jia Zhang ◽  
Meng Zhou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Novel Approach ◽  
Porcine Embryo ◽  
Development And Differentiation ◽  
Distinct Signaling Pathway

The inner cell mass of the pre-implantation blastocyst consists of the epiblast and hypoblast from which embryonic stem cells (ESCs) and extra-embryonic endoderm (XEN) stem cells, respectively, can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of its in vivo tissue origin. We have developed a novel approach for deriving porcine XEN (pXEN) cells via culturing the blastocysts with a chemical cocktail culture system. The pXEN cells were positive for XEN markers, including Gata4, Gata6, Sox17, and Sall4, but not for pluripotent markers Oct4, Sox2, and Nanog. The pXEN cells also retained the ability to undergo visceral endoderm (VE) and parietal endoderm (PE) differentiation in vitro. The maintenance of pXEN required FGF/MEK+TGFβ signaling pathways. The pXEN cells showed a stable phenotype through more than 50 passages in culture and could be established repeatedly from blastocysts or converted from the naïve-like ESCs established in our lab. These cells provide a new tool for exploring the pathways of porcine embryo development and differentiation and providing further reference to the establishment of porcine ESCs with potency of germline chimerism and gamete development.

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