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.

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.

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.

scholarly journals Epigenetic control of embryonic stem cell fate

2010 ◽  
Vol 207 (11) ◽  
pp. 2287-2295 ◽  
Author(s):  
Nicolaj Strøyer Christophersen ◽  
Kristian Helin
Keyword(s):  
Cell Fate ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Types ◽  
Culture Conditions ◽  
Polycomb Group ◽  
Es Cell ◽  
Defined Culture

Embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and are pluripotent, as they are able to differentiate into all cell types of the adult organism. Once established, the pluripotent ES cells can be maintained under defined culture conditions, but can also be induced rapidly to differentiate. Maintaining this balance of stability versus plasticity is a challenge, and extensive studies in recent years have focused on understanding the contributions of transcription factors and epigenetic enzymes to the “stemness” properties of these cells. Identifying the molecular switches that regulate ES cell self-renewal versus differentiation can provide insights into the nature of the pluripotent state and enhance the potential use of these cells in therapeutic applications. Here, we review the latest models for how changes in chromatin methylation can modulate ES cell fate, focusing on two major repressive pathways, Polycomb group (PcG) repressive complexes and promoter DNA methylation.

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.

309 DEVELOPMENT OF A CULTURE SYSTEM CAPABLE OF LONG-TERM MAINTENANCE OF BUFFALO (BUBALUS BUBALIS) EMBRYONIC STEM CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 251 ◽  
Author(s):  
R. Sharma ◽  
A. George ◽  
N. M. Kamble ◽  
K. P. Singh ◽  
S. K. Panda ◽  
...  
Keyword(s):  
Colony Size ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Formation Rate ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Bubalus Bubalis ◽  
Es Cell

The present study was aimed at developing a system for long-term culture of buffalo embryonic stem (ES) cells, which, to our knowledge, have not been maintained beyond passage 10 in reports available to date, primarily because of lack of information on their specific requirements during in vitro culture. Inner cell mass (n = 181) cells, mechanically isolated from in vitro produced day 8 blastocysts, were cultured on mitomycin-C-treated buffalo fetal fibroblast feeder layers in stem cell medium (SCM), which consisted of Knockout-DMEM® + 15% Knockout serum replacer® + 1% minimal essential medium nonessential amino acids + 50 μg mL–1 of gentamicin, supplemented with 1000 IU mL–1 of leukemia inhibitory factor (LIF) and fibroblast growth factor-2 (FGF-2) at different concentrations. The medium was changed every 24 h. The primary colony formation rate, which was similar for 5, 10, 20, and 40 ng mL–1 of FGF-2 (63.7 ± 5.2, 65.7 ± 6.5, 57.0 ± 10.5, and 62.8 ± 13.30, respectively), was significantly higher (P ≤ 0.05) than that of controls (22.4 ± 5.5). In Experiment 2, ES-cell-like cell colonies at passages 6 through 7 (n = 441) were cultured for 5 to 6 days to examine the effects of media supplements. The percentage of colonies that survived was significantly higher (P ≤ 0.05) when these were cultured in SCM+LIF+5 ng mL–1 of FGF-2 (93.1 ± 1.8) than when these were cultured in SCM alone (73.5 ± 9.0) or in SCM supplemented with FGF-2 (88.8 ± 5.4) or LIF (85.8 ± 3.7). Following examination of the colony size at 0 and 120 h of culture, the increase in colony size was found to be nearly 4- (P ≤ 0.01) and 2-fold higher (P ≤ 0.05) with SCM+LIF+5 ng mL–1 of FGF-2 (41.9 ± 3.4) and SCM+FGF-2 (21.0 ± 3.0), respectively, than with SCM alone (10.8 ± 2.6) or with SCM+LIF (9.3 ± 3.3). The ES cell colonies cultured in the presence of FGF-2 were compact and had defined edges, whereas those cultured in its absence were less compact, irregularly shaped, and had less defined edges. To confirm the role of FGF-2 in maintenance of buffalo ES cells, the cell colonies cultured in the presence of 5 ng mL–1 of FGF-2 (n = 487) were exposed to different concentrations (10, 20, or 30 μM) of SU5402, a FGF-2 receptor inhibitor, for 5 to 6 days. The percentage of cell colonies that were found to have differentiated was significantly higher (P ≤ 0.05) when these had been cultured in the presence of 30 (78.6 ± 4.2) or 20 μM (47.9 ± 1.0) than when these were cultured with 10 (24.5 ± 5.1) or 0 μM (28.6 ± 2.3) of SU5402. Following culture in SCM+LIF+FGF-2, buffalo ES cells, in which the expression of pluripotency markers such as OCT-4, NANOG, and SOX-2 was regularly confirmed, have been maintained for more than 80 passages for over an year’s time to date, indicating that a combination of LIF and FGF-2 is beneficial for the maintenance of buffalo ES cells. Supported by NAIP grant No. C4/C-2067 from ICAR, India.

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.

Use of F9 teratocarcinoma STEM cells to study cell-matrix interactions in the early mouse embryo

1992 ◽  
Vol 50 (1) ◽  
pp. 602-603
Author(s):  
Marc Lenburg ◽  
Rulang Jiang ◽  
Lengya Cheng ◽  
Laura Grabel
Keyword(s):  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Embryoid Bodies ◽  
F9 Cells ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Cell Matrix Interactions ◽  
F9 Teratocarcinoma

We are interested in defining the cell-cell and cell-matrix interactions that help direct the differentiation of extraembryonic endoderm in the peri-implantation mouse embryo. At the blastocyst stage the mouse embryo consists of an outer layer of trophectoderm surrounding the fluid-filled blastocoel cavity and an eccentrically located inner cell mass. On the free surface of the inner cell mass, facing the blastocoel cavity, a layer of primitive endoderm forms. Primitive endoderm then generates two distinct cell types; parietal endoderm (PE) which migrates along the inner surface of the trophectoderm and secretes large amounts of basement membrane components as well as tissue-type plasminogen activator (tPA), and visceral endoderm (VE), a columnar epithelial layer characterized by tight junctions, microvilli, and the synthesis and secretion of α-fetoprotein. As these events occur after implantation, we have turned to the F9 teratocarcinoma system as an in vitro model for examining the differentiation of these cell types. When F9 cells are treated in monolayer with retinoic acid plus cyclic-AMP, they differentiate into PE. In contrast, when F9 cells are treated in suspension with retinoic acid, they form embryoid bodies (EBs) which consist of an outer layer of VE and an inner core of undifferentiated stem cells. In addition, we have established that when VE containing embryoid bodies are plated on a fibronectin coated substrate, PE migrates onto the matrix and this interaction is inhibited by RGDS as well as antibodies directed against the β1 integrin subunit. This transition is accompanied by a significant increase in the level of tPA in the PE cells. Thus, the outgrowth system provides a spatially appropriate model for studying the differentiation and migration of PE from a VE precursor.

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.

Sign in / Sign up

Export Citation Format

Share Document