405. Isolation and preliminary characterisation of putative sheep embryonic stem cells

2008 ◽  
Vol 20 (9) ◽  
pp. 85
Author(s):  
B. M. Murray ◽  
C. M. O.'Brien ◽  
J. L. Johnson ◽  
B. J. Conley ◽  
P. Bello ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Animal Health ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Well Being ◽  
Reproductive Technologies ◽  
Undifferentiated State

The establishment of true, fully characterised embryonic stem (ES) cells from livestock, (eg sheep) has yet to be reported. Such cells could make a significant impact on assisted reproductive technologies, vaccine delivery, and animal health and well being in the livestock industries. To date in sheep, there is a single report of putative ES cells which were maintained in an undifferentiated state for only 2 passages. Here we report the isolation and culture of pluripotent ES-like cells from in vivo derived, vitrified, sheep blastocysts. The inner cell mass of blastocysts were isolated by immunosurgery, cultured in Stem Cell Sciences' (SCS) novel inhibitor-based media, on a feeder layer of mouse embryonic fibroblasts (MEFs), resulting in putative ovine ES cells proliferating to at least passage 5. One cell line, BMCOV002, was established out of four thaw-recovered embryos. The colonies formed compact, near homogenous, small cell, multilayered, and well defined dome shaped masses that were morphologically similar to both mouse and human ES cell colonies. A peripheral halo of filamentous differentiated cells was detected in selected colonies from passage 3 to passage 5. The putative ovine ES-like cells were passaged by mechanical excision between days 6–7, and these colonies stained positive for alkaline phosphatase at both passage 3 and passage 5. Expression levels of genes encoding the pluripotent transcription factors OCT4, SOX2, REX1 and NANOG are shown using RT PCR in cells from passage 3. An important first step in studying the properties of ovine ES-like cells is the ability shown here to isolate and culture cells. Our attention now is focussed on maintaining these cells for some months in an undifferentiated state, and on being able to successfully cryopreserve and regenerate these 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.

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).

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.

scholarly journals Derivation, growth and applications of human embryonic stem cells

Reproduction ◽  
2004 ◽  
Vol 128 (3) ◽  
pp. 259-267 ◽  
Author(s):  
Miodrag Stojkovic ◽  
Majlinda Lako ◽  
Tom Strachan ◽  
Alison Murdoch
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Normal Karyotype ◽  
Medical Application ◽  
Stem Cell Markers ◽  
Undifferentiated State ◽  
Hes Cells

Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass cells of blastocysts with the potential to maintain an undifferentiated state indefinitely. Fully characterised hES cell lines express typical stem cell markers, possess high levels of telomerase activity, show normal karyotype and have the potential to differentiate into numerous cell types under in vitro and in vivo conditions. Therefore, hES cells are potentially valuable for the development of cell transplantation therapies for the treatment of various human diseases. However, there are a number of factors which may limit the medical application of hES cells: (a) continuous culture of hES cells in an undifferentiated state requires the presence of feeder layers and animal-based ingredients which incurs a risk of cross-transfer of pathogens; (b) hES cells demonstrate high genomic instability and non-predictable differentiation after long-term growth; and (c) differentiated hES cells express molecules which could cause immune rejection. In this review we summarise recent progress in the derivation and growth of undifferentiated hES cells and their differentiated progeny, and the problems associated with these techniques. We also examine the potential use of the therapeutic cloning technique to derive isogenic hES cells.

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.

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

185 THE PORCINE EPIBLAST AND NOT THE INNER CELL MASS HAS DEVELOPED CONVENTIONAL PATHWAYS FOR REGULATION OF PLURIPOTENCY

2009 ◽  
Vol 21 (1) ◽  
pp. 191
Author(s):  
V. J. Hall ◽  
J. Christensen ◽  
P. Maddox-Hyttel
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Positive Control ◽  
Total Rna ◽  
Weak Expression ◽  
Whole Transcriptome

Pluripotency in mice and human embryonic stem cells is regulated by a number of transcription factors, notably including Oct-4, Sox-2, and Nanog. However, in the pig, previous research indicates that Oct-4 protein and mRNA is not specifically localized to the inner cell mass (ICM) of the zona-intact (ZI) blastocyst. Levels of expression of Nanog mRNA, on the other hand, appear to be low in the ZI blastocyst, and protein has not been detected. Similarly, Sox-2 expression in the ZI blastocyst is relatively low and not specific to the ICM. In this study, we investigated the mRNA expression of Oct-4, Sox-2, and Nanog in D6/D7-derived ZI porcine in vivo-derived blastocysts compared with epiblasts mechanically isolated from hatched D10/D11 in vivo-derived blastocysts. We then investigated components involved in pathways important for regulating pluripotency, including JAK/STAT (i.e. gp130, LIFr), FGF (i.e. bFGF, FGFr1, FGFr2), and BMP (bmp4, smad4) signaling pathways and their downstream targets, stat3, c-myc, c-fos, by using RT-PCR. Sows were artificially inseminated, and embryos were flushed from uteri following slaughter. Single D6/D7 blastocysts (n = 3), single mechanically isolated D10/D11 epiblasts (n = 3), endometrium, and oviduct total RNA was isolated using the RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Total RNA from the blastocysts and epiblasts was then amplified to form cDNA using the QuantiTect Whole Transcriptome kit (Qiagen). Positive control tissues (oviduct and endometrium) were reverse transcribed using the RevertAid First Strand cDNA synthesis kit (Fermentas, Burlington, Ontario, Canada). Primers were designed to span introns in highly homologous sequences to human mRNA. Primers were tested in both oviduct and endometrium tissue, and products were sequenced to confirm specificity. PCR was performed at 55°C for 35 cycles. Results indicate that D6/D7 blastocysts only expressed Oct-4 and not Nanog and Sox-2. In contrast, all 3 transcripts were expressed in D10/D11 epiblasts. The D10/D11 epiblasts also expressed LIFr, bFGF, FGFr1, FGFr2, bmp4, smad4, stat3, c-myc, and c-fos. The cytokine receptor gp130 was only weakly expressed in a single epiblast. In contrast, the earlier stage D6/D7 blastocysts failed to express these messengers with the exception of weak expression of gp130 in all 3 blastocysts, and only a single blastocyst expressed LIFr, smad4, c-myc, and c-fos. In conclusion, this study indicates that the ICM of the porcine D6/D7 ZI blastocyst has not developed pluripotency signaling as observed in mice and humans at this developmental stage. Furthermore, without expression of gp130, the JAK/STAT pathway is unlikely to play a role in regulating pluripotency in the epiblast. It is likely that the later stage epiblast may be more amenable for the derivation of porcine embryonic stem cells.

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