Chromatin regulation landscape of embryonic stem cell identity

2010 ◽  
Vol 31 (2) ◽  
pp. 77-86 ◽  
Author(s):  
Yun Hwa Lee ◽  
Qiang Wu
Keyword(s):  
Stem Cell ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Histone Variants ◽  
Transcriptional Networks ◽  
Chromatin Regulation ◽  
Es Cell ◽  
Cell Identity

ES cells (embryonic stem cells) derived from the ICM (inner cell mass) of blastocysts are pluripotent and are capable of giving rise to most cell types. The ES cell identity is mainly maintained by the Oct4 (octamer-binding transcription factor 4) and Nanog transcriptional networks. Recently, a tremendous amount of work has focused on deciphering how ES cell identity is regulated epigenetically. It has been shown that histone methylation/demethylation, histone acetylation/deacetylation, histone variants and chromatin remodelling play crucial roles in ES cell maintenance and differentiation. Moreover, perturbation of those chromatin regulators results in loss of ES cell identity or aberrant differentiation. Therefore, it is important to fully understand the chromatin regulation landscape of ES cells. The knowledge gained will help us to harness the unique characteristics of ES cells for stem cell-related therapy and regenerative medicine. In the present review, we will discuss recent proceedings that provide novel insights into chromatin regulation of ES cell identity.

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.

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 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.

197 PRODUCTION OF IN VITRO- AND IN VIVO-DERIVED CAT BLASTOCYSTS FOR THE ESTABLISHMENT OF FELINE EMBRYONIC STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 207 ◽  
Author(s):  
J. Kehler ◽  
M. Roelke-Parker ◽  
B. Pukazhenthi ◽  
W. Swanson ◽  
C. Ware ◽  
...  
Keyword(s):  
Cell Lines ◽  
Chorionic Gonadotropin ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Es Cell ◽  
Feeder Layers

Identification and characterization of spontaneously occurring genetic diseases in cats has permitted the development of valuable models for testing potential treatments of similar human diseases. With the near completion of the feline genome project, establishment of pluripotential feline embryonic stem (ES) cells would facilitate the targeting of specific genetic loci to produce new feline medical models. Two approaches were used to produce feline blastocysts in an attempt to establish feline ES cells in culture. Naive queens were superovulated with an intramuscular (i.m.) injection of 150 IU of equine chorionic gonadotropin (eCG) followed by an i.m. injection of 100 IU of human chorionic gonadotropin (hCG) 80 h later; follicles were aspirated laparoscopically 24-26 h later for subsequent in vitro fertilization (IVF). On average, 29 mature cumulus oocyte cell complexes (COCs) were recovered from each queen. IVF was performed in 50 microliter drops of complete Hams F-10 medium containing 30 000 fresh, motile sperm. COCs were cultured overnight in 5% carbon dioxide at 38�C, and residual adherent cumulus cells were removed 12 to 16 h later by trituration in 0.1% hyaluronidase. Embryos were cultured in fresh drops of Hams F-10, and on average 25% developed to the early blastocyst stage after 7 days. Alternatively, estrus was induced in queens with a single i.m. injection of 100 IU of eCG, and then 72 h later queens were permitted six supervised matings with a fertile tom over the next two days. Queens underwent ovariohysterectomy 7 days after their first copulation, and compacted morulae and early blastocysts were flushed from the oviducts and uterine horns. On average, eight embryos were recovered from the reproductive tract of each queen. Both in vivo- and in vitro-matured blastocysts were subsequently cultured in standard mouse ES cell medium on inactivated mouse embryonic fibroblasts. When they failed to hatch in culture after 3 days, a 0.5% pronase solution was used to dissolve the zonae pellucidae under microscopic visualization. Denuded expanded blastocysts adhered to the heterotypic feeder layer and primary inner cell mass (ICM) outgrowths formed within 4 days. Outgrowths were mechanically disaggregated into small clusters of 15 to 20 cells and re-plated on fresh feeders. These colonies grew slowly and were transferred after one week onto new feeder layers. The addition of murine or human recombinant leukemia inhibitory factor had no effect on the survival and proliferation of primary outgrowths or subsequent colonies. After 3 weeks, all colonies derived from both in vivo- and in vitro-matured blastocysts had either differentiated or died. Additional experiments are ongoing to test the effects of homotypic feeder layers and alternative growth factors on promoting the establishment and survival of feline ES cell lines. Ultimately, germline transmission of any putative feline ES cell lines will need to be demonstrated in vivo for their utility in gene targeting experiments to be realized.

232 SERUM-FREE DERIVATION OF CAT EMBRYONIC STEM-LIKE CELLS DERIVED FROM IN VIVO-PRODUCED BLASTOCYSTS ON CAT EMBRYONIC FIBROBLAST FEEDERS

2007 ◽  
Vol 19 (1) ◽  
pp. 232 ◽  
Author(s):  
X. F. Yu ◽  
X. J. Jin ◽  
D. S. Choi ◽  
D. K. Jung ◽  
B. H. Choi ◽  
...  
Keyword(s):  
Culture System ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Es Cell ◽  
Serum Free ◽  
Embryonic Fibroblast

Fetal bovine serum (FBS) has usually been used as a component of the medium for the culture of embryonic stem (ES) cells. However, FBS contains undefined factors, which promote cell proliferation and occasionally stimulate differentiation of ES cells. To establish an effective culture system for the derivation and maintenance of cat ES cells, we compared the effects of KnockoutTM Serum Replacement (Invitrogen, Seoul, South Korea)-supplemented medium (KSR-medium) and FBS-supplemented medium (FBS-medium) on the attachment and formation of primary ES-like cell colonies of isolated inner cell mass (ICM) derived from in vivo-produced blastocysts on mitotically inactivated cat embryonic fibroblast (CEF) feeders. An inseminated domestic female cat underwent ovariohysterectomy at Day 7 post-insemination, after which blastocysts were flushed from the uterine horns. Inner cell mass (ICM) was mechanically isolated from the in vivo-matured blastocysts (n = 22) and cultured in KSR-medium or FBS-medium on a CEF feeder layer at 39�C, 5% CO2 in air. KSR-medium or FBS-medium consisted of knockoutTM DMEM supplemented with 20% KSR or FBS, 100 IU mL-1 penicillin/streptomycin, 2 mM l-glutamine, 1� MEM non-essential amino acids, 100 �M �-mercaptoethanol, and 1000 IU mL-1 recombinant mouse LIF. The results indicate that incidence of primary ICM attachment (91.6 � 14.4 vs. 55.5 � 9.6% in KSR- and FBS-medium, respectively; P < 0.05) and ES-like cell colony formation (63.8 � 12.7 vs. 27.7 � 4.8% in KSR- and FBS-medium, respectively; P < 0.05) is significantly affected by the ES cell culture medium. However, cat ES-like cells in KSR-medium did not actively proliferate in the primary culture. The number of cat ES-like cells in a colony after 5 days of culture was significantly higher in FBS-medium than in KSR-medium (749 � 82 vs. 383 � 55% in FBS- and KSR-medium, respectively; P < 0.05). A total of 10 ES-like cell colonies were formed in KSR-medium (n = 7) and FBS-medium (n = 3), and these expressed high levels of alkaline phosphatase activity (AP). Immunostaining analysis was positive for the ES cell-markers, Oct-4 and SSEA-3. To our knowledge, this is the first reported isolation of cat ES-like cells on CEF feeders in the absence of serum. A culture system of embryo-derived stem cells using serum-free medium may make it possible to directly examine the effects of factors added to culture medium on isolation and maintenance of cat ES cell lines in vitro. This work was supported by KOSEF (grant # M105250100 01-05N2501-00110).

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.

297 DERIVATION AND CHARACTERIZATION OF THE TRANSGENIC SOMATIC CELL NUCLEAR TRANSFER-DERIVED BOVINE EMBRYONIC STEM CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 246
Author(s):  
S. H. Jeong ◽  
H. S. Kim ◽  
H. Lee ◽  
K. J. Uh ◽  
S. H. Hyun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Mass Culture ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Es Cell ◽  
Trophoblast Cells

Bovine transgenic embryonic stem (ES) cells have not been reported yet because it seems that the derivation methods and the culture conditions for the inner cell mass are neither consistent nor optimized. Isolation of inner cell mass and primary culture of ES colonies is a critical step toward the establishment of authentic bovine ES cell lines. Herein, we reconstructed somatic cell nuclear transferred (SCNT) bovine blastocysts carrying a vector expressing the human INF-α gene, and isolated inner cell masses to derive transgenic bovine embryonic stem cells. In addition, we added 2 inhibitors, inhibition (2i system) of the mitogen-activated protein kinase (Erk1/2) cascade, PD0325901(3 Î1/4M), and of glycogen synthase kinase 3, CHIR99021 (1 Î1/4M), in the inner cell mass primary culture to check reliability of the 2i system for bovine ES culture. The 2 inhibitors made the morphology of colonies more intact, and primary colonies were better maintained in early passages. However, there were no significant effects on the attachment rate and maintenance in late passages (percent of percent over 3 passages: 2i system, 21/38 (55.3%); control, 22/42 (33.3%); P < 0.05). Inner cell masses were isolated mechanically and subcultured by an enzymatic in primary inner cell mass culture. Massive growth of trophoblast cells appears to inhibit inner cell mass growth, so hatching and hatched blastocysts were cut with a needle to remove trophoblast cells. Poor quality blastocysts were attached by the whole seeding method, and the margin trophoblast cells were consecutively removed in early passages. Established bovine ES cells express alkaline phosphatase, Oct-4, SSEA1, SSEA4, Tra-1–60, and Tra-1–81. We confirmed pluripotent gene expression of bovine ES like cells; Oct-4, SSEA1, and Rex 1 were positive, but trophoblast marker CDX2 was negative. This study shows that the 2i system is a reasonable method for use during inner cell mass culture in early passages. We established 6 transgenic nuclear transfer bovine ES cell lines with the 2i system and 4 in vitro fertilized bovine ES cell lines (all were over 10 passages).

scholarly journals Lack of beta 1 integrin gene in embryonic stem cells affects morphology, adhesion, and migration but not integration into the inner cell mass of blastocysts.

1995 ◽  
Vol 128 (5) ◽  
pp. 979-988 ◽  
Author(s):  
R Fässler ◽  
M Pfaff ◽  
J Murphy ◽  
A A Noegel ◽  
S Johansson ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Junctions ◽  
Es Cell ◽  
Beta 1 Integrin ◽  
Integrin Gene ◽  
And Migration

A gene trap-type targeting vector was designed to inactivate the beta 1 integrin gene in embryonic stem (ES) cells. Using this vector more than 50% of the ES cell clones acquired a disruption in the beta 1 integrin gene and a single clone was mutated in both alleles. The homozygous mutant did not produce beta 1 integrin mRNA or protein, while alpha 3, alpha 5, and alpha 6 integrin subunits were transcribed but not detectable on the cell surface. Heterozygous mutants showed reduced beta 1 expression and surface localization of alpha/beta 1 heterodimers. The alpha V subunit expression was not impaired on any of the mutants. Homozygous ES cell mutants lacked adhesiveness for laminin and fibronectin but not for vitronectin and showed a reduced association with a fibroblast feeder layer. Furthermore, they did not migrate towards chemoattractants in fibroblast medium. None of these functions were impaired in heterozygous mutants. Scanning electron microscopy revealed that homozygous cells showed fewer cell-cell junctions and had many microvilli not usually found on wild type and heterozygous cells. This profound change in cell shape is not associated with gross alterations in the expression and distribution of cytoskeletal components. Unexpectedly, microinjection into blastocysts demonstrated full integration of homozygous and heterozygous mutants into the inner cell mass. This will allow studies of the consequences of beta 1 integrin deficiency in several in vivo situations.

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