scholarly journals 181 Oct-4: A POTENTIAL MARKER FOR PLURIPOTENCY IN CATTLE

2005 ◽  
Vol 17 (2) ◽  
pp. 241
Author(s):  
M. Vejlsted ◽  
B. Avery ◽  
J. Gjoerret ◽  
M. Schmidt ◽  
T. Greve ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Bovine Embryo ◽  
Feeder Cells ◽  
Paraffin Sections ◽  
Specific Staining ◽  
Potential Marker

The POU (Pit-Oct-Unc) domain transcription factor Oct-4 is one of the most acknowledged markers for pluripotency in murine and primate embryonic cells. At the blastocyst stage, expression of Oct-4 has been shown to remain high in the inner cell mass (ICM) while being rapidly down regulated in the trophectoderm (TE). Furthermore, in these species, expression of Oct-4 is maintained in pluripotent derivatives of the ICM and in embryonic stem (ES) cells, but lost upon differentiation. In the bovine embryo, a marker with similar qualities has long been sought. The aim of this study was to investigate, using a commercially available antibody for immunohistochemistry (IHC), whether Oct-4 might serve this role. In vitro produced (IVP) embryos were transferred to synchronized recipients at Day 6 post insemination (p.i) and flushed at Day 12; in vivo-derived embryos were flushed at Day 14. Day 8 IVP embryos (n = 20) were fixed and processed for IHC in paraffin sections together with Day 12 (n = 18) and Day 14 (n = 3) embryos. From Day 8 IVP embryos, outgrowth colonies (OCs) were formed by intact blastocyst culture on mouse SNL feeder cells. OCs were photographed using a stereomicroscope on Days 12, 14, and 16 p.i., and were examined for Oct-4 expression by in situ IHC at Day 16 p.i. (n = 94). From isolated embryonic discs of Day 12 embryos, OCs were derived by similar culture and were either processed for IHC on paraffin sections at Days 16, 18, and 20 p.i. (n = 9) or used for establishment of ES-like cell lines. Of colonies formed, representative specimens from each of the initial 5 passages (n = 18) were examined for Oct-4 expression either in paraffin sections or in situ. In Day 8 embryos, Oct-4 expression was demonstrated in all nuclei of both ICM and TE cells except for presumptive apoptotic ones. Approximately one-fifth of the OCs presented a substantial amount of Oct-4 positive cells of putative ICM, but also of TE origin. Apparently, the formation of Oct-4 positive OCs was favored by initial attachment of the embryonic pole to the feeder cells. In Day 12 and 14 embryos, specific and exclusive Oct-4 staining of nuclei of the complete epiblast, but not the hypoblast and the TE, was revealed. All OCs derived from Day 12 embryonic discs showed specific staining for Oct-4 in nuclei of putative epiblast origin only. On subsequent culture of these isolated epiblast derivatives, loss of Oct-4 staining from colonies was observed by passage 3. This study has, for the first time, shown expression of Oct-4 to be limited to pluripotent cells of bovine Day 12 and 14 embryos. Compared with murine and primate embryos, down-regulation of Oct-4 expression in bovine TE cells appears to be delayed. Findings indicate that Oct-4 may be used as a marker for pluripotency in bovine ES-like cells, although TE derivatives may maintain Oct-4 expression when isolated from Day 8 embryos.

scholarly journals 192ISOLATION AND CULTURE OF EMBRYONIC STEM CELL-LIKE CELLS FROM IN VITRO FERTILIZED PORCINE BLASTOCYSTS

2004 ◽  
Vol 16 (2) ◽  
pp. 217
Author(s):  
H.-Y. Son ◽  
C.-H. Park ◽  
S.-G. Lee ◽  
G.-S. Lee ◽  
H.-S. Kim ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Feeder Cells ◽  
Es Cell ◽  
Transgenic Pigs ◽  
Murine Embryonic Fibroblast

The establishment of porcine embryonic stem (ES) cell lines should be useful for the production of transgenic pigs and studies of developmental gene regulation. Recent development of techniques for production of embryos in vitro could be a useful source for the isolation of ES cells. Therefore, to establish porcine ES cells, this study was conducted to isolate and culture inner cell mass (ICM) from in vitro-fertilized (IVF) porcine blastocysts. Cumulus-oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Oocytes were then fertilized using a modified swim-up method to prevent polyspermy and cultured to the blastocyst stage. Initial culture of ICM was conducted after either culture of whole embryos or isolation of ICM by immunosurgery. Developing IVF embryos were continuously cultured in 50% DMEM and 50% F-10 with 15% fetal bovine serum, 1% non-essential amino acids, 1.7mM L-glutamine, 1% penicillin/streptomycin, 0.1mM α-mercaptoethanol, 1000 unit recombinant human LIF, 40ngmL−1 recombinant human SCF and 20ngmL−1 recombinant human basic FGF on a mytomycin-C-inactivated murine embryonic fibroblast (MEF) feeder layer. Antibodies against porcine cells were produced in rabbit. After removal of zona pellucida, ICMs were isolated by immunosurgery and cultured on feeder cells the same as described above. After IVF, the rates of 2-cell embryos and blastocysts were 70.8% and 20.4%, respectively. Results from the isolation and culture of ICMs of porcine blastocysts are shown in following table. ICM isolated by immunosurgery showed better attachment to feeder cells and ES cell colony formation than cultured whole blastocysts. Morphology of colonies was similar to that of mouse ES cells, showing compact colonies with delineated boundary. Also, these colonies showed alkaline phosphatase activity. Porcine ES-cell like colonies were passed 3 times through physical separation on fresh feeder layers. These results indicated that porcine ES-like cell line can be established from IVF porcine blastocysts. Further characterization of these porcine ES-like cell lines is required. Table 1 Isolation and culture of ICM from porcine blastocyst produced by IVF

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

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.

183 DEVELOPMENT OF PORCINE EMBRYOS MICROINJECTED WITH PORCINE EMBRYONIC GERM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 199
Author(s):  
C.-H. Park ◽  
S.-G. Lee ◽  
D.-H. Choi ◽  
M.-G. Kim ◽  
C. K. Lee
Keyword(s):  
Germ Cells ◽  
Fluorescent Protein ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Green Fluorescent Protein Gene ◽  
Cleavage Stage ◽  
Allocation Pattern

Embryonic germ (EG) cells, derived from primordial germ cells in the developing fetus, are similar to embryonic stem (ES) cells in terms of expression pattern of undifferentiated markers and their ability to colonize both the somatic and the germ cell lines following injection into a host blastocyst, which has been proven in mouse. Several studies using porcine EG cells have shown that it is possible to produce somatic chimeras after blastocyst injection. However, not only was the degree of reported chimerism low, but also there has been no report about the fate of injected EG cells in porcine blastocysts. This study was designed to observe the distribution pattern of porcine EG cells in chimeric blastocyst after injection into cleavage-stage porcine embryos. To ascertain development of microinjected porcine embryos with EG cells, 10 to 15 EG cells were injected into cleavage stage of in vitro fertilized embryos and cultured up to blastocyst. Also, porcine EG cells were labeled with DiO (Invitrogen, Carlsbad, CA) on the cell membrane or transfected with green fluorescent protein gene to observe whether the EG cells injected in the host embryo would incorporate into the inner cell mass (ICM) or trophectoderm (TE). Chimeric embryos were produced and allowed to develop into blastocysts to investigate the injected EG cells would come to lie in ICM and/or TE of the blastocyst, by scoring their position. In result, developmental rate was similar in all treatments. In all treatments, EG cells were mainly allocated in both ICM and TE of the chimeric blastocysts. These results suggest that examining the allocation pattern of injected EG cells, maintained pluripotency in vitro, could provide clues of differentiation process in vivo. Furthermore, to enhance the allocation of EG cells into the embryonic lineage, it would be required to optimize the culture condition for EG cells as well as embryos. Further experiment are needed to determine whether the injected EG cells could maintain their properties throughout the environment in the embryonic development in vitro. Table 1. Distribution of the porcine EG cells microinjected into cleavage-stage embryos

287 ACTIVE MITOCHONDRIA ARE PRESENT IN MOUSE AND MONKEY EMBRYONIC STEM CELL LINES

2008 ◽  
Vol 20 (1) ◽  
pp. 223 ◽  
Author(s):  
T. Lonergan ◽  
A. Harvey ◽  
J. Zhao ◽  
B. Bavister ◽  
C. Brenner
Keyword(s):  
Cell Lines ◽  
Complex I ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Mouse Fibroblast ◽  
Es Cell ◽  
Cell Content ◽  
Stem Cell Lines

The inner cell mass (ICM) of the blastocyst develops into the fetus after uterine implantation. Prior to implantation, ICM cells synthesize ATP by glycolytic reactions. We now report that cells of the ICM in 3.5-day-old mouse embryos have too few mitochondria to be visualized with either Mitotracker red (active mitochondria) or an antibody against complex I of OXPHOS. By comparison, all of the surrounding trophectoderm cells reveal numerous mitochondria throughout their cytoplasm. It has largely been assumed that embryonic stem (ES) stem cells derived from the ICM also have few mitochondria, and that replication of mitochondria in the ES cells does not begin until they commence differentiation. We further report that mouse E14 ES cells and monkey ORMES 7 ES cells have considerable numbers of active mitochondria when cultured under standard conditions, i.e., 5% CO2 in air. Both the mouse E14 and monkey ES cell lines expressed two markers of undifferentiated cells, Oct-4 and SSEA-4, and monkey ES cells expressed the undifferentiated cell marker Nanog; however, Oct-4 is nonspecific in monkey ES cells because trophectoderm also expresses this marker, unlike in mice. Ninety-nine percent of the E14 cells examined, and 100% of the ORMES 7 cells, have a visible mitochondrial mass when stained with either Mitoracker red or with an antibody against OXPHOS complex I. The ATP content in the mouse E14 cells (4.13 pmoles ATP/cell) is not significantly different (P = 0.76) from that in a mouse fibroblast control (3.75 pmoles ATP/cell). Cells of the monkey ORMES 7 cell line had 61% of the ATP/cell content (7.55 pmoles ATP/cell) compared to the monkey fibroblast control (12.38 pmoles ATP/cell). Both cell lines expressed two proteins believed to indicate competence of mitochondria to replicate: PolG, the polymerase used to replicate the mitochondrial genome, and TFAM, a nuclear-encoded transcription factor reported to regulate several aspects of mitochondrial function. Both proteins were found to co-localize in the mitochondria. We conclude that when the ICMs are isolated from blastocysts and used to establish these two ES cell lines in cell culture, mitochondrial biosynthesis is activated.

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.

Human embryonic stem cells: prototypical pluripotential progenitors

2002 ◽  
Vol 10 (3) ◽  
pp. 187-199 ◽  
Author(s):  
R Mollard ◽  
BJ Conley ◽  
AO Trounson
Keyword(s):  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Germ Layers ◽  
Mouse Es Cells ◽  
Morphological Criteria ◽  
Human Es Cells

Embryonic stem (ES) cells are a primitive cell type derived from the inner cell mass (ICM) of the developing embryo. When cultured for extended periods, ES cells maintain a high telomerase activity, normal karyotype and the pluripotential developmental capacity of their ICM derivatives. Such capacity is best demonstrated by mouse ES cells which can contribute to all tissues of the developing embryo following either their injection into host blastocysts or tetraploid embryo complimentation (for a review see Robertson). For both practical and ethical reasons it is not possible to inject human ES cells into blastocysts for the development of a term fetus. However, when injected beneath the testicular capsule of severe combined immunodeficient (SCID) mice, human ES cells form teratomas comprising tissue representatives of all three embryonic germ layers (ectoderm, mesoderm and endoderm) thus attesting to their pluripotency. Based upon morphological criteria, neuronal, cardiac, bone, squamous epithelium, skeletal muscle, gut and respiratory epithelia are readily identifiable within the human ES-cell-derived teratomas. With the demonstrated capability to isolate and maintain pluripotent human ES cells in vitro, their ability to give rise to tissue representatives of all three embryonic germ layers and the technical advances made possible by research on mouse ES cells, a rapid increase in human ES cell research aimed at drug discovery and human cell and gene therapies has occurred. Indeed in the mouse, dissociated embryoid bodies (EBs) have already been demonstrated capable of repopulating the haematopoietic system of recipient animals (for a review see Keller) and mouse ES cells are currently being used in attempts to repair mouse neural degenerative lesions.

Establishment of rat embryonic stem-like cells from the morula using a combination of feeder layers

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 229-237 ◽  
Author(s):  
Chiaki Sano ◽  
Asako Matsumoto ◽  
Eimei Sato ◽  
Emiko Fukui ◽  
Midori Yoshizawa ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Preimplantation Embryos ◽  
Long Term Culture ◽  
Oct4 Expression ◽  
Feeder Layers

SummaryEmbryonic stem (ES) cells are characterized by pluripotency, in particular the ability to form a germline on injection into blastocysts. Despite numerous attempts, ES cell lines derived from rat embryos have not yet been established. The reason for this is unclear, although certain intrinsic biological differences among species and/or strains have been reported. Herein, using Wistar-Imamichi rats, specific characteristics of preimplantation embryos are described. At the blastocyst stage, Oct4 (also called Pou5f1) was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was localized to the TE. In contrast, at an earlier stage, expression of Oct4 was detected in all the nuclei in the morula. These stages were examined using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging) to establish rat ES-like cell lines. The rat ES-like cell lines obtained from the morula maintained expression of Oct4 over long-term culture, whereas cell lines derived from blastocysts lost pluripotency during early passage. The morula-derived ES-like cell lines showed Oct4 expression in a long-term culture, even after cryogenic preservation, thawing and EGFP transfection. These results indicate that rat ES-like cell lines with long-term Oct4 expression can be established from the morula of Wistar-Imamichi rats using a combination of feeder layers.

Generation of embryonic stem cells: limitations of and alternatives to inner cell mass harvest

2008 ◽  
Vol 24 (3-4) ◽  
pp. E4 ◽  
Author(s):  
Sunit Das ◽  
Michael Bonaguidi ◽  
Kenji Muro ◽  
John A. Kessler
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Ethical Considerations ◽  
Pluripotent Cells ◽  
Mammalian Embryo ◽  
Induced Pluripotency ◽  
Early Mammalian Embryo

✓ Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the early mammalian embryo. Because of their plasticity and potentially unlimited capacity for self-renewal, ES cells have generated tremendous interest both as models for developmental biology and as possible tools for regenerative medicine. This excitement has been attenuated, however, by scientific, political, and ethical considerations. In this article the authors describe somatic cell nuclear transfer and transcription-induced pluripotency, 2 techniques that have been used in attempts to circumvent the need to derive ES cells by the harvest of embryonic tissue.

W41/W41 blastocyst complementation: a system for genetic modeling of hematopoiesis

Blood ◽  
2010 ◽  
Vol 115 (1) ◽  
pp. 47-50 ◽  
Author(s):  
Lina Jansson ◽  
Jonas Larsson
Keyword(s):  
Inner Cell Mass ◽  
Fetal Liver ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Mouse Strains ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Blastocyst Complementation

Abstract We report a rapid and highly efficient approach to generate mice in which the hematopoietic system is derived from embryonic stem (ES) cells. We show that ES cells injected into blastocysts from the c-kit–deficient W41/W41 mouse strain have a clear advantage over the W41/W41 blastocyst-derived inner cell mass cells in founding the hematopoietic system. Fetal liver hematopoietic stem cells from W41/W41 blastocyst complementation embryos can be transplanted to establish large cohorts of bone marrow chimeras with hematopoiesis of practically pure ES-cell origin. Using ES cells with site-directed modifications, we show how this system can be used to drive inducible transgene expression in hematopoietic cells in a robust and reliable manner both in vitro and in vivo. The approach avoids the cost and time constraints associated with the creation of standard transgenic mouse strains while taking advantage of the sophisticated site-directed manipulations that are possible in ES cells.

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