Embryo implantation and embryonic stem cell development in primates

2001 ◽  
Vol 13 (8) ◽  
pp. 517 ◽  
Author(s):  
John P. Hearn
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Embryo Implantation ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Passive Immunization ◽  
Embryo Viability ◽  
Successful Establishment

The endocrine dialogue that results in implantation and the successful establishment of pregnancy in primates relies on embryonic secretion of chorionic gonadotrophin (CG). This hormone is a signal of embryo viability and capacity to support the corpus luteum. The expression of CG is apparently restricted to primates. Active or passive immunization of marmoset monkeys against the beta subunit of CG prevented implantation and early pregnancy, without disrupting the ovarian cycle. Studies of individual embryos cultured in vitro showed that CG is secreted at low levels by the blastocyst from before attachment, with secretion increasing exponentially after attachment. Gonadotrophin releasing hormone (GnRH) was also secreted, from mid-blastocyst stages, before the detection of CG. The secretion of GnRH by the embryo continued through the attachment and outgrowth stages of embryonic differentiation in vitro. The hypothetical role of GnRH in regulating CG release during implantation was tested in recently completed experiments. Individual embryos cultured with GnRH, or with agonist or antagonist to GnRH, showed significant variations in their secretion of CG and in their survival in culture, suggesting a causal relationship between these hormones. Embryos cultured with natural GnRH showed enhanced growth and development. Embryonic stem cells, from the inner cell mass of marmoset and rhesus monkeys, were the first primate embryonic stem cells to be isolated and characterized, enabling the subsequent isolation of human embryonic stem 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 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.

scholarly journals Network based meta-analysis prediction of microenvironmental relays involved in stemness of human embryonic stem cells

2014 ◽  
Author(s):  
Virginie Mournetas ◽  
Quentin M. Nunes ◽  
Patricia A. Murray ◽  
Christopher M. Sanderson ◽  
David G. Fernig
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Meta Analysis ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Interaction Network ◽  
Transcriptional Networks

Background. Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of in vitro fertilised blastocysts, which can either be maintained in an undifferentiated state or committed into lineages under determined culture conditions. These cells offer great potential for regenerative medicine, but at present, little is known about the mechanisms that regulate hESC stemness; in particular, the role of cell-cell and cell-extracellular matrix interactions remain relatively unexplored. Methods and results. In this study we have performed an in silico analysis of cell-microenvironment interactions to identify novel proteins that may be responsible for the maintenance of hESC stemness. A hESC transcriptome of 8,934 mRNAs was assembled using a meta-analysis approach combining the analysis of microarrays and the use of databases for annotation. The STRING database was utilised to construct a protein-protein interaction network focused on extracellular and transcription factor components contained within the assembled transcriptome. This interactome was structurally studied and filtered to identify a short list of 92 candidate proteins, which may regulate hESC stemness. Conclusion. We hypothesise that this list of proteins, either connecting extracellular components with transcriptional networks, or with hub or bottleneck properties, may contain proteins likely to be involved in determining stemness.

scholarly journals Absence of Basement Membranes after Targeting the LAMC1 Gene Results in Embryonic Lethality Due to Failure of Endoderm Differentiation

1999 ◽  
Vol 144 (1) ◽  
pp. 151-160 ◽  
Author(s):  
Neil Smyth ◽  
H. Seda Vatansever ◽  
Patricia Murray ◽  
Michael Meyer ◽  
Christian Frie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Basement Membrane ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Basement Membranes ◽  
Embryoid Bodies ◽  
Endoderm Differentiation

The LAMC1 gene coding for the laminin γ1 subunit was targeted by homologous recombination in mouse embryonic stem cells. Mice heterozygous for the mutation had a normal phenotype and were fertile, whereas homozygous mutant embryos did not survive beyond day 5.5 post coitum. These embryos lacked basement membranes and although the blastocysts had expanded, primitive endoderm cells remained in the inner cell mass, and the parietal yolk sac did not develop. Cultured embryonic stem cells appeared normal after targeting both LAMC1 genes, but the embryoid bodies derived from them also lacked basement membranes, having disorganized extracellular deposits of the basement membrane proteins collagen IV and perlecan, and the cells failed to differentiate into stable myotubes. Secretion of the linking protein nidogen and a truncated laminin α1 subunit did occur, but these were not deposited in the extracellular matrix. These results show that the laminin γ1 subunit is necessary for laminin assembly and that laminin is in turn essential for the organization of other basement membrane components in vivo and in vitro. Surprisingly, basement membranes are not necessary for the formation of the first epithelium to develop during embryogenesis, but first become required for extra embryonic endoderm differentiation.

scholarly journals Network based meta-analysis prediction of microenvironmental relays involved in stemness of human embryonic stem cells

2014 ◽  
Author(s):  
Virginie Mournetas ◽  
Quentin M. Nunes ◽  
Patricia A. Murray ◽  
Christopher M. Sanderson ◽  
David G. Fernig
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Meta Analysis ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Interaction Network ◽  
Transcriptional Networks

Background. Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of in vitro fertilised blastocysts, which can either be maintained in an undifferentiated state or committed into lineages under determined culture conditions. These cells offer great potential for regenerative medicine, but at present, little is known about the mechanisms that regulate hESC stemness; in particular, the role of cell-cell and cell-extracellular matrix interactions remain relatively unexplored. Methods and results. In this study we have performed an in silico analysis of cell-microenvironment interactions to identify novel proteins that may be responsible for the maintenance of hESC stemness. A hESC transcriptome of 8,934 mRNAs was assembled using a meta-analysis approach combining the analysis of microarrays and the use of databases for annotation. The STRING database was utilised to construct a protein-protein interaction network focused on extracellular and transcription factor components contained within the assembled transcriptome. This interactome was structurally studied and filtered to identify a short list of 92 candidate proteins, which may regulate hESC stemness. Conclusion. We hypothesise that this list of proteins, either connecting extracellular components with transcriptional networks, or with hub or bottleneck properties, may contain proteins likely to be involved in determining stemness.

scholarly journals Network based meta-analysis prediction of microenvironmental relays involved in stemness of human embryonic stem cells

2014 ◽  
Author(s):  
Virginie Mournetas ◽  
Quentin M. Nunes ◽  
Patricia A. Murray ◽  
Christopher M. Sanderson ◽  
David G. Fernig
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Meta Analysis ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Interaction Network ◽  
Transcriptional Networks

Background. Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of in vitro fertilised blastocysts, which can either be maintained in an undifferentiated state or committed into lineages under determined culture conditions. These cells offer great potential for regenerative medicine, but at present, little is known about the mechanisms that regulate hESC stemness; in particular, the role of cell-cell and cell-extracellular matrix interactions remain relatively unexplored. Methods and results. In this study we have performed an in silico analysis of cell-microenvironment interactions to identify novel proteins that may be responsible for the maintenance of hESC stemness. A hESC transcriptome of 8,934 mRNAs was assembled using a meta-analysis approach combining the analysis of microarrays and the use of databases for annotation. The STRING database was utilised to construct a protein-protein interaction network focused on extracellular and transcription factor components contained within the assembled transcriptome. This interactome was structurally studied and filtered to identify a short list of 92 candidate proteins, which may regulate hESC stemness. Conclusion. We hypothesise that this list of proteins, either connecting extracellular components with transcriptional networks, or with hub or bottleneck properties, may contain proteins likely to be involved in determining stemness.

389 CULTURE OF EQUINE INNER CELL MASS USING EQUINE MESENCHYMAL CELLS AS A FEEDER LAYER

2010 ◽  
Vol 22 (1) ◽  
pp. 351
Author(s):  
J. F. Lima-Neto ◽  
G. H. M. Araujo ◽  
M. L. Alvarenga ◽  
C. F. Moya-Araujo ◽  
I. D. P. Blanco ◽  
...  
Keyword(s):  
Amino Acids ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Embryoid Body ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Feeder Layer ◽  
Current Experiment

Stem cells can originate from embryo, fetus, and adults tissues. Adult stem cells are present in almost all organs and are responsible for tissue regeneration and repair. The mesenchymal stem cells (MSC) are multipotent cells present in the bone marrow. The plasticity of these cells allows them to be used in cell therapy because they have the potential to differentiate into several tissues with mesenchymal origin. On the other hand, embryonic stem cells (ESC) have the ability to differentiate into the 3 embryonic tissues (endoderm, mesoderm, and ectoderm) generating all kinds of tissue in the living organism. Embryonic stem cells can be maintained in their undifferentiated state when cultured in vitro in the presence of the leukemia inhibitory factor (LIF) and over a feeder layer composed normally by mouse fibroblasts. Because of the small number of articles studying equine ESC, the current experiment aimed to compare the utilization of equine fibroblasts and equine MSC as feeders for inner cell mass (ICM) culture. Eighteen blastocysts were collected on Day 7 after ovulation from 5 fertile mares that had been artificially inseminated. The embryos were placed in a petri dish with Dulbecco’s phosphate buffered saline + 10% FCS, and the ICM was mechanically removed using 2 insulin needles. The ICM was then transferred to one well of 24 well plates containing either an equine fibroblast monolayer (9 embryos) or a MSC monolayer (9 embryos). Both feeders layers were inactivated with 10 μg mL-1 mitomycine C, and the cell concentration was 140 000 cells/well. The culture media utilized was the DMEM/F12 with 20% FCS, 1% essential amino acids, 1% nonessential amino acids, 0.1 mM β-mercaptoetanol, 10 ng mL-1 LIF, penicillin, streptomycin, and amphotericin B. The ICM was cultured during 5 days for the adherence to the plate, and the media was change every 2 days. Data was analyzed by ANOVA. Results indicated no significant differences between the use of equine fibroblasts or MSC as feeder layer to support equine ICM in culture. The expansion of the ICM cells was observed after 10 to 12 days in culture in 44.4% (4/9) of ICM cultured in MSC monolayer and 55.5% (5/9) of the ICM cultured over a fibroblast layer. The formation of embryoid body-like structures were observed after the second passage (5-7 days in culture) in 50% (2/4) of embryos using MSC feeders and in 40% of embryos (2/5) for which fibroblasts were used. Cultures were then positively marked with the Oct 4 antibody for characterization of the undifferentiated lineage. The results of the current experiment showed that although further patronization of the culture system is still needed, both fibroblasts and MSC are suitable as feeders for the culture of equine ICM when aiming to establish ESC lineage in vitro. Financial support was provided by FAPESP and CNPq.

scholarly journals Differentiation of Human Embryonic Stem Cells into Neuron, Cholinergic, and Glial Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Kimia Hosseini ◽  
Emilia Lekholm ◽  
Aikeremu Ahemaiti ◽  
Robert Fredriksson
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Glial Cells ◽  
Human Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cholinergic Neurons ◽  
Neuronal Cultures ◽  
Short Period

Human embryonic stem cells (hESCs) are pluripotent cells, capable of differentiation into different cellular lineages given the opportunity. Derived from the inner cell mass of blastocysts in early embryonic development, the cell self-renewal ability makes them a great tool for regenerative medicine, and there are different protocols available for maintaining hESCs in their undifferentiated state. In addition, protocols for differentiation into functional human neural stem cells (hNSCs), which have the potential for further differentiation into various neural cell types, are available. However, many protocols are time-consuming and complex and do not always fit for purpose. In this study, we carefully combined, optimized, and developed protocols for differentiation of hESCs into adherent monolayer hNSCs over a short period of time, with the possibility of both expansion and freezing. Moreover, the method details further differentiation into neurons, cholinergic neurons, and glial cells in a simple, single step by step protocol. We performed immunocytochemistry, qPCR, and electrophysiology to examine the expression profile and characteristics of the cells to verify cell lineage. Using presented protocols, the creation of neuronal cultures, cholinergic neurons, and a mixed culture of astrocytes and oligodendrocytes can be completed within a three-week time period.

2. Embryonic stem cells

2021 ◽  
pp. 21-37
Author(s):  
Jonathan Slack
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Practical Importance ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Human Embryos ◽  
Mouse Es Cells ◽  
Stage Embryo

‘Embryonic stem cells’ focuses on embryonic stem (ES) cells, which are grown in tissue culture from the inner cell mass of a mammalian blastocyst-stage embryo. Human ES cells offer a potential route to making the kinds of cells needed for cell therapy. ES cells were originally prepared from mouse embryos. Although somewhat different, cells grown from inner cell masses of human embryos share many properties with mouse ES cells, such as being able to grow without limit and to generate differentiated cell types. Mouse ES cells have so far been of greater practical importance than those of humans because they have enabled a substantial research industry based on the creation of genetically modified mice.

216 EMBRYONIC AND INDUCED PLURIPOTENT STEM CELLS ANALOGOUS TO INNER CELL MASS-DERIVED LIF-DEPENDENT MOUSE EMBRYONIC STEM CELLS ESTABLISHED FROM THE DOMESTIC PIG, SUS SCROFA

2012 ◽  
Vol 24 (1) ◽  
pp. 220
Author(s):  
B. P. Telugu ◽  
T. Ezashi ◽  
A. Alexenko ◽  
S. Lee ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Cell Lines ◽  
Umbilical Cord ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Induced Pluripotent

Authentic embryonic stem cells (ESC) may never have been successfully derived from the inner cell mass (ICM) of pig and other ungulates, despite over 25 years of effort. Recently, porcine induced pluripotent stem cells (piPSC) were generated by reprogramming somatic cells with a combination of four factors OCT4, SOX2, KLF4 and c-MYC (OSKM) delivered by lentiviral transduction. The established piPSC are analogous to FGF2-dependent human (h) ESC and murine “epiblast stem cells,” and are likely to advance swine as a model in biomedical research. Here, we report for the first time, the establishment of LIF-dependent, so called naïve type pluripotent stem cells (1) from the inner cell mass (ICM) of porcine blastocysts by up-regulating the expression of KLF4 and POU5F1; and (2) from umbilical cord mesenchyme (Wharton's jelly) by transduction with OSKM factors and subsequent culture in the presence of LIF-based medium with inhibitors that substitute for low endogenous expression of c-MYC and KLF4 and promote pluripotency. The 2 compounds that have been used in this study are, CHIR99021 (CH), which substitutes c-MYC by inhibiting GSK3B and activating WNT signalling and Kenpaullone (KP), which inhibits both GSK3B and CDK1 and supplants KLF4 function. The lentiviral vectors employed for introducing the re-programming genes were modified for doxycycline-mediated induction of expression (tet-on) and are ‘floxed’ for Cre-mediated recombination and removal of transgenes following complete reprogramming. Two LIF-dependent cell lines have been derived from the ICM cells of late d 5.5 in vitro produced blastocysts and four from umbilical cord mesenchyme recovered from fetuses at d 35 of pregnancy. The derived stem cell lines are alkaline phosphatase-positive, resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile and expression of pluripotent markers, such as POU5F1, SOX2 and surface marker SSEA1. They are dependent on LIF signalling for maintenance of pluripotency, can be cultured over extended passage (>50) with no senescence. Of importance, the ICM-derived lines have been successful in their ability to form teratomas. The cells could be cultured in feeder free conditions on a synthetic matrix in the presence of chemically defined medium and can be coaxed to differentiate under xeno-free conditions. Currently, the piPSC lines are being investigated for their ability to give rise to teratomas and to produce a live offspring by nuclear transfer. Supported by Addgene Innovation Award, MO Life Sciences Board Grant 00022147 and NIH grant HD21896.

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