2 BOVINE EMBRYONIC STEM-LIKE CELLS DERIVED FROM IN VITRO-PRODUCED BLASTOCYSTS

2017 ◽  
Vol 29 (1) ◽  
pp. 108
Author(s):  
Y. S. Bogliotti ◽  
J. Wu ◽  
M. Vilariño ◽  
K. Suzuki ◽  
J. C. Belmonte ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Basal Medium ◽  
Primitive Endoderm ◽  
Culture Dish ◽  
Rock Inhibitor ◽  
Immunodeficient Mice

Embryonic stem cells (ESC) are derived from the inner cell mass (ICM) of preimplantation blastocysts. To date, it has been challenging to establish pluripotent ESC lines for domestic animals, which could be important for biotechnological applications, such as genetic engineering and SCNT, and biomedical research. The aim of this work was to derive and characterise bovine embryonic stem-like cells (bESC) from in vitro-produced bovine blastocysts. Embryos were produced by in vitro fertilization of in vitro-matured oocytes aspirated from abattoir ovaries and cultured in groups of 25 in 50-μL drops of KSOM (Evolve, Zenith Biotech) with 4 mg mL−1 BSA for 7 days until they reached the blastocyst stage (Ross et al., 2009 Reproduction 137, 427–437). At that point, the zona pellucida (ZP) was removed using 1 mg mL−1 Pronase (Sigma, St. Louis, MO), and ZP-free blastocysts were washed 6 times in SOF-HEPES. Three derivation approaches were tested: ZP-free whole blastocysts, mechanically isolated ICM, and immunosurgery-derived ICM. In each case, individual blastocysts/ICM were placed in 1 well of a 12-well dish seeded with a monolayer of mouse embryo fibroblasts (MEF) and cultured in mTeSR1 basal medium (without growth factors) supplemented with 20 ng mL−1 FGF2 and 2.5 μM IWR1 (CTFR) (Wu et al. 2015 Nature 521, 316–321). After 48 h, blastocysts/ICM that failed to adhere were physically pressed against the bottom of the culture dish with a 22-gauge needle under a stereoscope to aid attachment. Thereafter, the media was changed daily. Outgrowths (after 6–7 days in culture) were dissociated and passaged using TrypLE and re-seeded in the presence of ROCK inhibitor (Y-27632, 10 μM) onto newly prepared wells containing MEF. Established bESC lines were cultured on MEF and passaged every 4 to 5 days at a 1:10 split ratio. The bESC lines were characterised by immunofluorescence (IF), RNA-seq, and teratoma formation. The efficiency of cell line derivation (evaluated at passage 3) was similar for the 3 approaches: whole blastocysts (9/16, 56.3%), mechanical ICM isolation (7/12, 58.3%), and immunosurgical ICM isolation (7/16, 43.8%). The bESC were passaged and cultured long-term (more than 15 passages) and were subjected to several rounds of freezing and thawing while retaining their morphology and characteristics. IF analysis showed that long-term cultured bESC expressed the markers SOX2 and OCT4 (pluripotency), but did not express CDX2 (trophectoderm) or GATA6 (primitive endoderm). RNAseq analysis of 2 bESC lines showed that ICM markers (POU5F1, NANOG, SOX2, LIN28B, DNAMT3B, UTF1, SALL4) were expressed (RPKM > 0.4), while trophectoderm markers (CDX2, GATA2, GATA3, FGF4, TFAP2A) and primitive endoderm markers (GATA6, HNF4A) were not expressed (RPKM < 0.4). Finally, bESC lines (n = 2) were able to form teratomas in immunodeficient mice. The teratomas contained tissues representative of the 3 germ lineages and expressed lineage-specific markers (ectoderm: TUJ1, endoderm: FOXA2, and mesoderm: ASM). In conclusion, the culture condition used in this work (CTFR) enables robust derivation and long-term in vitro propagation of pluripotent bESC.

scholarly journals The birth of embryonic pluripotency

2014 ◽  
Vol 369 (1657) ◽  
pp. 20130541 ◽  
Author(s):  
Thorsten Boroviak ◽  
Jennifer Nichols
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Positional Information ◽  
Eutherian Mammal ◽  
Primitive Endoderm ◽  
Early Cleavage ◽  
Fgf Signalling

Formation of a eutherian mammal requires concurrent establishment of embryonic and extraembryonic lineages. The functions of the trophectoderm and primitive endoderm are to enable implantation in the maternal uterus, axis specification and delivery of nutrients. The pluripotent epiblast represents the founding cell population of the embryo proper, which is protected from ectopic and premature differentiation until it is required to respond to inductive cues to form the fetus. While positional information plays a major role in specifying the trophoblast lineage, segregation of primitive endoderm from epiblast depends upon gradual acquisition of transcriptional identity, directed but not initiated by fibroblast growth factor (FGF) signalling. Following early cleavage divisions and formation of the blastocyst, cells of the inner cell mass lose totipotency. Developing epiblast cells transiently attain the state of naive pluripotency and competence to self-renew in vitro as embryonic stem cells and in vivo by means of diapause. This property is lost after implantation as the epiblast epithelializes and becomes primed in preparation for gastrulation and subsequent organogenesis.

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.

scholarly journals Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marino Maemura ◽  
Hiroaki Taketsuru ◽  
Yuki Nakajima ◽  
Ruiqi Shao ◽  
Ayaka Kakihara ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Primitive Endoderm ◽  
Cell Stage ◽  
Supportive Environment ◽  
Mouse Zygotes ◽  
Single Blastomeres ◽  
Multicellular Organisms

AbstractIn multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

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.

scholarly journals Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations

Reproduction ◽  
2015 ◽  
Vol 150 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Young-Ho Choi ◽  
Pablo Ross ◽  
Isabel C Velez ◽  
B Macías-García ◽  
Fernando L Riera ◽  
...  
Keyword(s):  
High Glucose ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Primitive Endoderm ◽  
Blastocyst Development ◽  
Embryo Culture Medium ◽  
Effect Of Glucose

Equine embryos developin vitroin the presence of high glucose concentrations, but little is known about their requirements for development. We evaluated the effect of glucose concentrations in medium on blastocyst development after ICSI. In experiment 1, there were no significant differences in rates of blastocyst formation among embryos cultured in our standard medium (DMEM/F-12), which contained >16 mM glucose, and those cultured in a minimal-glucose embryo culture medium (<1 mM; Global medium, GB), with either 0 added glucose for the first 5 days, then 20 mM (0-20) or 20 mM for the entire culture period (20-20). In experiment 2, there were no significant differences in the rates of blastocyst development (31–46%) for embryos cultured in four glucose treatments in GB (0-10, 0-20, 5-10, or 5-20). Blastocysts were evaluated by immunofluorescence for lineage-specific markers. All cells stained positively forPOU5F1. An inner cluster of cells was identified that included presumptive primitive endoderm cells (GATA6-positive) and presumptive epiblast (EPI) cells. The 5-20 treatment resulted in a significantly lower number of presumptive EPI-lineage cells than the 0-20 treatment did.GATA6-positive cells appeared to be allocated to the primitive endoderm independent of the formation of an inner cell mass, as was previously hypothesized for equine embryos. These data demonstrate that equine blastocyst development is not dependent on high glucose concentrations during early culture; rather, environmental glucose may affect cell allocation. They also present the first analysis of cell lineage allocation inin vitro-fertilized equine blastocysts. These findings expand our understanding of the factors that affect embryo development in the horse.

Leptin treatment of in vitro cultured embryos increases outgrowth rate of inner cell mass during embryonic stem cell derivation

2019 ◽  
Vol 55 (7) ◽  
pp. 473-481 ◽  
Author(s):  
Ali Cihan Taskin ◽  
Ahmet Kocabay ◽  
Ayyub Ebrahimi ◽  
Sercin Karahuseyinoglu ◽  
Gizem Nur Sahin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Stem Cell Derivation

Derivation of human embryonic stem cell lines after blastocyst microsurgery

2010 ◽  
Vol 88 (3) ◽  
pp. 479-490 ◽  
Author(s):  
Guoliang Meng ◽  
Shiying Liu ◽  
Xiangyun Li ◽  
Roman Krawetz ◽  
Derrick E. Rancourt
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Clinical Medicine ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Hesc Lines

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. Because of their ability to differentiate into a variety of cell types, human embryonic stem cells (hESCs) provide an unlimited source of cells for clinical medicine and have begun to be used in clinical trials. Presently, although several hundred hESC lines are available in the word, only few have been widely used in basic and applied research. More and more hESC lines with differing genetic backgrounds are required for establishing a bank of hESCs. Here, we report the first Canadian hESC lines to be generated from cryopreserved embryos and we discuss how we navigated through the Canadian regulatory process. The cryopreserved human zygotes used in this study were cultured to the blastocyst stage, and used to isolate ICM via microsurgery. Unlike previous microsurgery methods, which use specialized glass or steel needles, our method conveniently uses syringe needles for the isolation of ICM and subsequent hESC lines. ICM were cultured on MEF feeders in medium containing FBS or serum replacer (SR). Resulting outgrowths were isolated, cut into several cell clumps, and transferred onto fresh feeders. After more than 30 passages, the two hESC lines established using this method exhibited normal morphology, karyotype, and growth rate. Moreover, they stained positively for a variety of pluripotency markers and could be differentiated both in vitro and in vivo. Both cell lines could be maintained under a variety of culture conditions, including xeno-free conditions we have previously described. We suggest that this microsurgical approach may be conducive to deriving xeno-free hESC lines when outgrown on xeno-free human foreskin fibroblast feeders.

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

165 EXPRESSION OF Oct-4 IN BUFFALO (BUBALUS BUBALIS) EMBRYOS GENERATED THROUGH IN VITRO FERTILIZATION OR PARTHENOGENETIC ACTIVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 162
Author(s):  
D. Kumar ◽  
T. Anand ◽  
K. P. Singh ◽  
M. S. Chauhan ◽  
P. Palta ◽  
...  
Keyword(s):  
Relative Humidity ◽  
Inner Cell Mass ◽  
Polar Body ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Bubalus Bubalis ◽  
Dnase I ◽  
Parthenogenetic Activation ◽  
Cell Lysate

Octamer-4 (Oct-4) is a member of Class V of the POU transcription factors family, which is involved in transcriptional regulation during early embryonic development and cell differentiation. It is expressed in the inner cell mass of blastocysts and in embryonic stem cells (ESCs), and its expression is widely used as a marker of pluripotency in ESCs in many species. This study was, therefore, carried out to examine the expression of Oct-4 in embryos at the 2-, 4-, 8- to 16-cell, morula, and blastocyst stages generated through IVF or parthenogenetic activation. A total of 100 embryos were used in the study, 10 for each embryonic stage from both methods of embryo production. Immature oocytes obtained from slaughterhouse buffalo (Bubalus bubalis) ovaries were subjected to IVM in TCM-199 + 10% FBS + 5 µg mL–1 pFSH + 0.81 mm sodium pyruvate for 24 h in a CO2 incubator (5% O2, 5% CO2, 90–95% relative humidity) at 38.5�C. IVF was carried out immediately after IVM; the cleaved embryos were cultured for 8 days in CR2 medium containing 0.6% BSA and 10% FBS for production of embryos at different stages. For production of embryos through parthenogenesis, after 24 h of IVM, oocytes were denuded of cumulus cells by incubation in 0.2% hyaluronidase in Dulbecco's phosphate-buffered saline for 2 min. The denuded oocytes with a prominent polar body were parthenogenetically activated by exposure to 7% ethanol for 7 min, followed by incubation with 2 mm 6-dimethyl aminopurine in CR2 medium for 3.5 h in a CO2 incubator (5% O2, 5% CO2, 90–95% relative humidity) at 38.5�C, and then subjected to IVC as described above. A two-step RT-PCR was carried out using Cells-to-cDNA Kit-II (Ambion, Austin, TX, USA), using bovine primers 52-GTT CTC TTT GGA AAG GTG TTC-3' and 5'-ACA CTC GGA CCA CGT CTT TC-3' for the amplification of Oct-4. For this, the embryos were washed with PBS, transferred to 30 µL of cold cell lysis buffer and incubated in a thermal cycler at 75�C for 10 min. The cell lysate was treated with DNase-I at 37�C for 30 min to degrade genomic DNA and then heated at 75�C for 5 min to inactivate the DNase-I. The cell lysate (10 µL) was used for making cDNA using random primer. The PCR cycle included heating to 94�C for 2 min, followed by 33 cycles of 94�C for 30 s, 57�C for 30 s, and 72�C for 45 s. A final extension at 72�C for 10 min was carried out to complete the amplification of the Oct-4 gene. Transcripts of Oct-4 were detected at all of the embryonic stages, from the 2-cell through the hatched blastocyst stage in both IVF and parthenogenetically generated embryos. These results indicate that Oct-4, which is believed to be a reliable marker for pluripotency of ESCs in a number of species, is expressed in early cleavage-stage buffalo embryos and continues to be expressed in preimplantation-stage blastocysts.

396 Oct-4 EXPRESSION IN CAT INNER CELL MASS-DERIVED CELLS CULTURED IN MEDIUM WITH DIFFERENT PROTEIN SOURCES

2010 ◽  
Vol 22 (1) ◽  
pp. 354
Author(s):  
T. S. Rascado ◽  
J. F. Lima-Neto ◽  
S. E. R. S. Lorena ◽  
B. W. Minto ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Domestic Cat ◽  
Santa Cruz

The domestic cat can be used as a biological model for humans because of similarities in some disease and genetically transmitted conditions. Embryonic stem cells might complete nuclear reprogramming more efficiently than somatic cells and, therefore, are potentially useful for increasing interspecific cloning success. The objective of this study was to establish an effective culture system for inner cell mass (ICM)-derived cells in the domestic cat, testing the ability of the ICM to attach to the culture dish and to form embryonic stem cell colonies in the presence of fetal calf serum (FCS) and Knockout serum (KS). Moreover, knowing that the transcription factor Oct-4 is important for the maintenance of pluripotency in human and murine embryonic stem cells, the expression of this factor was evaluated in in vitro-produced blastocyst and in the attached ICM. Domestic cat oocytes were matured, fertilized, and cultured in vitro until the blastocyst stage. The ICM was mechanically isolated (n = 60) using a scalpel blade and transferred to a monolayer of chemically inactivated cat fibroblasts with 10 μg mL-1 mitomicin C. The base culture media (BM) was DMEM/F12 supplemented with nonessential amino acids, glutamine, leukemia inhibitory factor, fibroblast growth factor-2, 2-mercaptoethanol, and antibiotics. Three groups were tested: G1 = BM with 20% FCS (20); G2 = BM with 20% KS (20); G3 = BM with 15% FSC and 5% KS (20). Culture was performed in a 5% CO2 in air incubator at 38.5°C. No statistical difference was observed among groups in relation to ICM attachment (chi-square, P > 0.05). Ninety percent of the ICM presented good adhesion after 3 days of culture and started to grow in all media tested. However, until now, no good colonies were formed. Fifteen blastocysts and 10 attached ICM were fixed in 3% paraformaldehyde and permeabilized in 0.2% triton X-100 in PBS. Subsequently, to block nonspecific binding of the primary antibody, the preadsorption for 2 h at room temperature with OCT4 blocking peptide (sc-8628P, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used. Samples were incubated with Oct4 antibody (N-19 : sc 8628, Santa Cruz Biotechnology) and with the appropriate secondary antibody (A21431, Invitrogen) and examined by fluorescence microscopy. Oct4 protein was detected both in the ICM and trophoderm cells, and it was distributed in cytoplasm and nuclei. These embryos were also stained with Hoechst 33342. Although further standardization of the culture media is needed, it seems that the KS can be replaced by FCS in cat embryonic stem cell culture. Furthermore, the immunostain of the trophoderm with Oct-4 indicates a difference in the expression of this factor when compared with its expression on human and murine blastocysts. This could be related to in vitro production, or Oct 4 is not a good pluripotency marker for cat embryos and cat embryonic stem cell, consequently. This fact has been noted in goat, bovine, and porcine embryos. Acknowledgment is given to FAPESP.

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