206 COMPARATIVE EVALUATION OF VARIOUS PROTOCOLS FOR NEURAL DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 211
Author(s):  
A. Tas ◽  
S. Arat ◽  
H. Dalcik
Keyword(s):  
Neuronal Differentiation ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Neuronal Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Dependent Manner ◽  
Neuronal Precursor Cells ◽  
Mesenchymal Differentiation

Mouse embryonic stem (ES) cells derived from the inner cell mass of blastocysts can differentiate into neuronal cells by treatment with retinoic acid (RA). ES cells cultured as aggregates and as single cell suspensions were then exposed to RA which induced multiple phenotypes of neuronal cells. Differentiation was dependent on the concentration of RA and the time of exposure. In this study, we cultured ES cells as a suspension in which they formed embryoid bodies (EBs). The EBs were treated with varying concentrations of RA for differing times. We used increasing concentrations of RA (50 nM, 100 nM, 1 �M, and 3 �M) prepared from a stock of 10 mM RA in DMSO. Immunocytochemistry staining was carried out on 2, 5, 7, and 9 days of culture. We formed EBs for 4 days with standard ES cell medium (without LIF) plus an additional 4 days of treatment with 1 �M RA. ES cells were treated with 1 �M RA for 2 days in suspension culture. Two-day-old EBs plated on culture dishes were treated with 1 �M RA for 3 days. To test for the effect of RA concentration on embryonic differentiation, 2-day-old EBs were treated with 50 nM, 100 nM, 1 �M, and 3 �M RA for 3 days. The time-dependent effects of RA on the 4-/4+ RA group were investigated. Results showed that neuronal precursor cells appeared on the second day of culture; they were stained with nestin antibody. On the 5th day of culture, neurons were detected with NCAM antibody. On the 7th day of culture, glial cells were observed with GFAP, and on the 9th day of culture GFAP, expression increased. In EBs that were plated and then treated with RA, the same results were obtained. RA induced neuronal differentiation in a concentration-dependent manner. Low concentrations (50 nM and 100 nM) of RA induced neuronal differentiation besides mesenchymal differentiation; however, higher concentrations (1 �M and 3 �M) of RA did not induce mesenchymal differentiation. The most efficient neuronal differentiation was obtained at 3 mM RA concentration. This study was performed in TUBITAK Research Institute for Genetic Engineering and biotechnology.

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.

228 ESTABLISHMENT OF EMBRYONIC STEM-LIKE CELL LINES IN RABBITS

2007 ◽  
Vol 19 (1) ◽  
pp. 230 ◽  
Author(s):  
Y.-W. Ou ◽  
K.-H. Lee ◽  
L.-R. Chen ◽  
P.-C. Tang ◽  
H.-F. Guu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Immunocytochemical Staining ◽  
Es Cell ◽  
Feeder Layers ◽  
Feeder Group

Embryonic stem (ES) cells are pluripotent cells from the inner cell mass (ICM) of the blastocyst. They are capable of differentiating to various cell types, such as neural cells, cardiocytes, hepatic cells, and germ cells. The aim of this study was to establish rabbit ES cell lines as an animal model for human diseases. Blastocysts were collected from New Zealand White rabbits during Days 4 to 5 after breeding. After removal of the mucin coat and the zona pellucida by pronase, the embryos were directly cultured in ES cell medium on mitomycin C-treated mouse embryonic fibroblast (MEF) or STO feeder layers. In Experiment 1, the efficiencies of 2 different feeder layers, MEF and STO, in generating rabbit ES cell lines were compared. Six blastocysts were used for each STO and MEF feeder group. The primary ICM colonies were formed in 67% (4/6) of the cultures on the STO and 83% (5/6) on the MEF. Sixty percent of those primary colonies (3/5) were successfully grown into ES-like cell lines in the MEF feeder group. However, no cell lines were established on the STO feeder. In Experiment 2, whole blastocysts or ICMs isolated by immunosurgery were cultured to establish ES cell lines. A total of 21 blastocysts were recovered from 2 does. Eighteen whole blastocysts and 3 isolated ICMs were cultured on the MEF feeders. Twelve (67%) of the cultured whole blastocysts formed primary ICM colonies, of which 5 (42%) of the cultures continuously propagated and formed ES-like cell lines. In the immunosurgical group, 2 of the 3 isolated ICMs formed primary colonies but only 1 ES-like cell line was established. A total of 9 ES-like cell lines maintained morphological undifferentiation after 14 passages and expressed alkaline phosphatase activity. Seven of the 9 ES-like cells expressed Oct-4 and the stage-specific embryonic antigen-4 (SSEA-4) as detected by immunocytochemical staining. Two cell lines were further induced to differentiate into embryoid bodies in suspension culture. Another 3 cell lines were injected into SCID mice and one of them formed a teratoma. The competence of generating chimeric rabbits and the teratogenicity of the established ES-like cell lines are under evaluation. In conclusion, rabbit ES-like cells were efficiently generated and whole-blastocyst culturing on the MEF feeder appeared to be a preferred method for the isolation and maintenance of rabbit ES-like cell lines.

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.

001. ISOLATION AND CHARACTERISATION OF PORCINE ES CELLS

2009 ◽  
Vol 21 (9) ◽  
pp. 1
Author(s):  
M. B. Nottle ◽  
I. M. Vassiliev ◽  
S. Vassilieva ◽  
L. F. S. Beebe ◽  
S. J. Harrison ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryoid Bodies ◽  
The Body ◽  
Domestic Species ◽  
Serum Replacement

Embryonic stem (ES) cellshave the capacity for self renewal, can remain undifferentiated in long term culture and can contribute to all the cells in the body including the germ cells. EScells have been isolated in mice and have also been described for humans. However despite considerable effort for more than two decades ES cellswhich can contribute to the germline are yet to be isolated for the pig or any domestic species for that matter. We have developed a new method for isolating porcine ES cells which uses whole embryos cultured in alpha MEM with 10% serum replacement plus additives under 5% O2. Unlike methods employed previously this method results in homogenous outgrowths whose cells resemble ES cells and which express Oct 4 and Nanog and SSEA-1 [1]. These cells can be passaged and cryopreserved repeatedly resulting in the establishment of cell lines at similar efficiencies to that reported previously for 129Sv mice [2]. These cells can form embryoid bodies and can be differentiated to various cell types representative of all three germ layers [3]. Following their injection into blastocysts these cells localise /become incorporated in the inner cell mass and can be used to produce chimaeras when these embryos are transferred to recipient animals [2]. To date we have produced chimaeric pigs from one male ES cell line [2]. These are currently being mated to demonstrate germline transmission. Future studies will examine the applicability of our method to other species commencing with mice and cattle before extending these to humans.

277 ESTABLISHMENT OF EMBRYONIC STEM CELL LINES DERIVED FROM A EGFP-TRANSGENIC MOUSE AND THEIR SURVIVAL IN THE COCHLEA OF C57BL/6 MICE

2008 ◽  
Vol 20 (1) ◽  
pp. 218
Author(s):  
K. S. Ahn ◽  
S. J. Jeon ◽  
J. Y. Jung ◽  
T. Choi ◽  
S. J. Choi ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Transgenic Mouse ◽  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Nerve Fibers ◽  
Embryoid Bodies ◽  
Ganglion Neurons

Embryonic stem (ES) cells isolated from inner cell mass cells of blastocyst-stage embryos are capable of differentiating into various cell lineages. Transplantation of these cells may potentially be a treatment for many degenerative diseases. Such cell therapy has often been tested using allografts of ES cells in mice. However, it has been difficult to locate transplanted ES cells and to avoid the rejection of allogeneic ES cells by the host. The aims of this study were to establish ES cell lines ubiquitously expressing enhanced green fluorescent protein (EGFP) and to test survival of ES cells in allografts into the cochlea of inbred C57BL/6 mice. Nine hatched blastocysts collected from a C57BL/6-green mouse that ubiquitously expresses transgene EGFP were plated onto an inactivated STO feeder layer. Two putative ES-like colonies were obtained from the plated blastocysts, and repeated subculture of these colonies produced two cell lines expressing EGFP. The cell lines possessed typical characteristics of ES cells, including densely packed colonies of the cells with prominent nucleoli, a high nuclear-cytoplasmic ratio, and high alkaline phosphatase activity. In suspension culture, these cells formed simple and cystic embryoid bodies. Undifferentiated EGFP-transgenic ES cells (106 cells per mouse) were injected into the cochlea of five C57BL/6 mice deafened by gentamycin treatment. Although no behavioral changes were noticed until four weeks after the transplantation, histological study revealed that grafted cells survived in the scala media of all injected mice. Incorporation of the cells expressing EGFP into the host was found along the auditory nerve fibers close to the organ of Corti. Such incorporation was also discovered in the area of the spiral ganglion neurons, cochlear sensory epithelia, and stria vascularis. Morphology and size of the cells varied depending on their sites of incorporation. The results from the present study demonstrate that, due to their survival in transplantation without allogeneic rejection as well as ubiquitous and stable expression of EGFP, ES cells from an EGFP-transgenic mouse may be a useful means of studying cell therapy.

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.

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