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.

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.

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.

396 Oct-4 EXPRESSION IN CAT INNER CELL MASS-DERIVED CELLS CULTURED IN MEDIUM WITH DIFFERENT PROTEIN SOURCES

2010 ◽  
Vol 22 (1) ◽  
pp. 354
Author(s):  
T. S. Rascado ◽  
J. F. Lima-Neto ◽  
S. E. R. S. Lorena ◽  
B. W. Minto ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Domestic Cat ◽  
Santa Cruz

The domestic cat can be used as a biological model for humans because of similarities in some disease and genetically transmitted conditions. Embryonic stem cells might complete nuclear reprogramming more efficiently than somatic cells and, therefore, are potentially useful for increasing interspecific cloning success. The objective of this study was to establish an effective culture system for inner cell mass (ICM)-derived cells in the domestic cat, testing the ability of the ICM to attach to the culture dish and to form embryonic stem cell colonies in the presence of fetal calf serum (FCS) and Knockout serum (KS). Moreover, knowing that the transcription factor Oct-4 is important for the maintenance of pluripotency in human and murine embryonic stem cells, the expression of this factor was evaluated in in vitro-produced blastocyst and in the attached ICM. Domestic cat oocytes were matured, fertilized, and cultured in vitro until the blastocyst stage. The ICM was mechanically isolated (n = 60) using a scalpel blade and transferred to a monolayer of chemically inactivated cat fibroblasts with 10 μg mL-1 mitomicin C. The base culture media (BM) was DMEM/F12 supplemented with nonessential amino acids, glutamine, leukemia inhibitory factor, fibroblast growth factor-2, 2-mercaptoethanol, and antibiotics. Three groups were tested: G1 = BM with 20% FCS (20); G2 = BM with 20% KS (20); G3 = BM with 15% FSC and 5% KS (20). Culture was performed in a 5% CO2 in air incubator at 38.5°C. No statistical difference was observed among groups in relation to ICM attachment (chi-square, P > 0.05). Ninety percent of the ICM presented good adhesion after 3 days of culture and started to grow in all media tested. However, until now, no good colonies were formed. Fifteen blastocysts and 10 attached ICM were fixed in 3% paraformaldehyde and permeabilized in 0.2% triton X-100 in PBS. Subsequently, to block nonspecific binding of the primary antibody, the preadsorption for 2 h at room temperature with OCT4 blocking peptide (sc-8628P, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used. Samples were incubated with Oct4 antibody (N-19 : sc 8628, Santa Cruz Biotechnology) and with the appropriate secondary antibody (A21431, Invitrogen) and examined by fluorescence microscopy. Oct4 protein was detected both in the ICM and trophoderm cells, and it was distributed in cytoplasm and nuclei. These embryos were also stained with Hoechst 33342. Although further standardization of the culture media is needed, it seems that the KS can be replaced by FCS in cat embryonic stem cell culture. Furthermore, the immunostain of the trophoderm with Oct-4 indicates a difference in the expression of this factor when compared with its expression on human and murine blastocysts. This could be related to in vitro production, or Oct 4 is not a good pluripotency marker for cat embryos and cat embryonic stem cell, consequently. This fact has been noted in goat, bovine, and porcine embryos. Acknowledgment is given to FAPESP.

scholarly journals Physical confinement signals regulate the organization of stem cells in three dimensions

2016 ◽  
Vol 13 (123) ◽  
pp. 20160613 ◽  
Author(s):  
Sebastian V. Hadjiantoniou ◽  
David Sean ◽  
Maxime Ignacio ◽  
Michel Godin ◽  
Gary W. Slater ◽  
...  
Keyword(s):  
Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Cell Interactions ◽  
Three Dimensions ◽  
Cell Substrate ◽  
Cell Cell

During embryogenesis, the spherical inner cell mass (ICM) proliferates in the confined environment of a blastocyst. Embryonic stem cells (ESCs) are derived from the ICM, and mimicking embryogenesis in vitro , mouse ESCs (mESCs) are often cultured in hanging droplets. This promotes the formation of a spheroid as the cells sediment and aggregate owing to increased physical confinement and cell–cell interactions. In contrast, mESCs form two-dimensional monolayers on flat substrates and it remains unclear if the difference in organization is owing to a lack of physical confinement or increased cell–substrate versus cell–cell interactions. Employing microfabricated substrates, we demonstrate that a single geometric degree of physical confinement on a surface can also initiate spherogenesis. Experiment and computation reveal that a balance between cell–cell and cell–substrate interactions finely controls the morphology and organization of mESC aggregates. Physical confinement is thus an important regulatory cue in the three-dimensional organization and morphogenesis of developing cells.

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.

scholarly journals mTOR Is Essential for Growth and Proliferation in Early Mouse Embryos and Embryonic Stem Cells

2004 ◽  
Vol 24 (15) ◽  
pp. 6710-6718 ◽  
Author(s):  
Mirei Murakami ◽  
Tomoko Ichisaka ◽  
Mitsuyo Maeda ◽  
Noriko Oshiro ◽  
Kenta Hara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Size ◽  
Inner Cell Mass ◽  
Critical Role ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Mouse Embryos ◽  
Early Mouse

ABSTRACT TOR is a serine-threonine kinase that was originally identified as a target of rapamycin in Saccharomyces cerevisiae and then found to be highly conserved among eukaryotes. In Drosophila melanogaster, inactivation of TOR or its substrate, S6 kinase, results in reduced cell size and embryonic lethality, indicating a critical role for the TOR pathway in cell growth control. However, the in vivo functions of mammalian TOR (mTOR) remain unclear. In this study, we disrupted the kinase domain of mouse mTOR by homologous recombination. While heterozygous mutant mice were normal and fertile, homozygous mutant embryos died shortly after implantation due to impaired cell proliferation in both embryonic and extraembryonic compartments. Homozygous blastocysts looked normal, but their inner cell mass and trophoblast failed to proliferate in vitro. Deletion of the C-terminal six amino acids of mTOR, which are essential for kinase activity, resulted in reduced cell size and proliferation arrest in embryonic stem cells. These data show that mTOR controls both cell size and proliferation in early mouse embryos and embryonic stem cells.

215 COMPARATIVE ANALYSIS OF PORCINE PLURIPOTENT CELL LINES DERIVED FROM SOMATIC CELLS AND VARIOUS EMBRYONIC ORIGINS

2012 ◽  
Vol 24 (1) ◽  
pp. 220
Author(s):  
J. K. Park ◽  
H. S. Kim ◽  
K. J. Uh ◽  
K. H. Choi ◽  
H. M. Kim ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Lines ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Pluripotent Cells ◽  
Differentiation Potential

Since pluripotent cells were first derived from the inner cell mass (ICM) of mouse blastocysts, tremendous efforts have been made to establish embryonic stem cell (ESC) lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to derive pluripotent cells of naïve state that represents full pluripotency, due to the frequent occurrence of spontaneous differentiation into an EpiSC-like state during culture in pigs. We have been able to derive EpiSC-like porcine embryonic stem cell (pESC) lines of a differentiated non-ES cell state from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF), in vivo derived, IVF aggregated and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) into porcine fibroblast cells. In this study, we analysed characteristics such as marker expression, pluripotency and the X chromosome inactivation (XCI) status of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1-60 and TRA 1-81. Furthermore all of these cell lines showed in vitro differentiation potential; female XCI activity and a normal karyotype. Here we provide preliminary results that suggest that, as a nonpermissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines. This work was supported by the BioGreen 21 Program (#20070401034031, PJ0081382011), Rural Development Administration, Republic of Korea.

scholarly journals The birth of embryonic pluripotency

2014 ◽  
Vol 369 (1657) ◽  
pp. 20130541 ◽  
Author(s):  
Thorsten Boroviak ◽  
Jennifer Nichols
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Positional Information ◽  
Eutherian Mammal ◽  
Primitive Endoderm ◽  
Early Cleavage ◽  
Fgf Signalling

Formation of a eutherian mammal requires concurrent establishment of embryonic and extraembryonic lineages. The functions of the trophectoderm and primitive endoderm are to enable implantation in the maternal uterus, axis specification and delivery of nutrients. The pluripotent epiblast represents the founding cell population of the embryo proper, which is protected from ectopic and premature differentiation until it is required to respond to inductive cues to form the fetus. While positional information plays a major role in specifying the trophoblast lineage, segregation of primitive endoderm from epiblast depends upon gradual acquisition of transcriptional identity, directed but not initiated by fibroblast growth factor (FGF) signalling. Following early cleavage divisions and formation of the blastocyst, cells of the inner cell mass lose totipotency. Developing epiblast cells transiently attain the state of naive pluripotency and competence to self-renew in vitro as embryonic stem cells and in vivo by means of diapause. This property is lost after implantation as the epiblast epithelializes and becomes primed in preparation for gastrulation and subsequent organogenesis.

scholarly journals Generation of embryonic stem cell lines from mouse blastocysts developed in vivo and in vitro: relation to Oct-4 expression

Reproduction ◽  
2006 ◽  
Vol 132 (1) ◽  
pp. 59-66 ◽  
Author(s):  
S Tielens ◽  
B Verhasselt ◽  
J Liu ◽  
M Dhont ◽  
J Van Der Elst ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Differentiation Potential ◽  
Stem Cell Lines

Embryonic stem (ES) cells are the source of all embryonic germ layer tissues. Oct-4 is essential for their pluripotency. Sincein vitroculture may influence Oct-4 expression, we investigated to what extent blastocysts culturedin vitrofrom the zygote stage are capable of expressing Oct-4 and generating ES cell lines. We comparedin vivowithin vitroderived blastocysts from B6D2 mice with regard to Oct-4 expression in inner cell mass (ICM) outgrowths and blastocysts. ES cells were characterized by immunostaining for alkaline phosphatase (ALP), stage-specific embryonic antigen-1 (SSEA-1) and Oct-4. Embryoid bodies were made to evaluate the ES cells’ differentiation potential. ICM outgrowths were immunostained for Oct-4 after 6 days in culture. A quantitative real-time PCR assay was performed on individual blastocysts. Of thein vitroderived blastocysts, 17% gave rise to ES cells vs 38% of thein vivoblastocysts. Six-day old outgrowths fromin vivodeveloped blastocysts expressed Oct-4 in 55% of the cases vs 31% of thein vitroderived blastocysts. The amount of Oct-4 mRNA was significantly higher for freshly collectedin vivoblastocysts compared toin vitrocultured blastocysts.In vitrocultured mouse blastocysts retain the capacity to express Oct-4 and to generate ES cells, be it to a lower level thanin vivoblastocysts.

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