Genomic imprinting in primate embryos and embryonic stem cells

2006 ◽  
Vol 18 (8) ◽  
pp. 817 ◽  
Author(s):  
Shoukhrat M. Mitalipov
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Paternal Allele ◽  
Aberrant Methylation ◽  
Es Cell ◽  
Biallelic Expression ◽  
Cell Based Therapy

Embryonic stem (ES) cells hold promise for cell and tissue replacement approaches to treating human diseases. However, long-term in vitro culture and manipulations of ES cells may adversely affect their epigenetic integrity including imprinting. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. We demonstrated aberrant biallelic expression of IGF2 and H19 in several rhesus monkey ES cell lines while SNRPN and NDN were normally imprinted and expressed from the paternal allele. In contrast, expanded blastocyst-stage embryos, from which these ES cells were derived, exhibited normal paternal expression of IGF2 and maternal expression of H19. To test the possibility that aberrant methylation at an imprinting centre (IC) upstream of H19 accounts for the relaxed imprinting of IGF2 and H19, we performed comprehensive methylation analysis by investigating methylation profiles of CpG sites within the IGF2/H19 IC. Our results demonstrate abnormal hypermethylation within the IGF2/H19 IC in all analysed ES cell lines consistent with biallelic expression of these genes. Cellular overproliferation and tumour formation resulting from tissue or cell transplantation are potential problems that must be addressed before clinical trials of ES cell-based therapy are initiated.

281 ATTEMPTS FOR ESTABLISHMENT OF PORCINE EMBRYONIC STEM-LIKE CELLS DERIVED FROM IN VITRO-PRODUCED BLASTOCYSTS

2009 ◽  
Vol 21 (1) ◽  
pp. 237
Author(s):  
H. M. Kim ◽  
J. K. Park ◽  
S. G. Lee ◽  
C. H. Park ◽  
S. W. Yoon ◽  
...  
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Control Group ◽  
Es Cell ◽  
Cell Stage

The porcine embryonic stem (ES) cells could be a useful tool for the production of transgenic animals and the study of developmental gene regulation. Even though the efficiency of establishment of ES cells from in vivo blastocysts is relatively high, especially in mice, it is difficult and expensive to obtain in vivo embryos in domestic animals. Recent development of techniques in the production of embryos in vitro could be a useful source for the establishment of ES cells. However, the morphology and cell quality of in vitro-produced embryos are inferior to those of their in vivo counterparts. Although many attempts have been made to establish ES cells from in vitro-produced embryos, the overall efficiency is extremely low because of the poor embryo quality. However, aggregation of in vitro-produced embryos was developed to increase the number of cells in the inner cell mass (ICM) of blastocysts and could be useful in the application to ES cell establishment. Therefore, in this study, we attempted to derive porcine ES cells by using aggregation of in vitro-produced embryos by in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). Cumulus–oocyte complexes were collected from prepubertal gilt ovaries and matured in vitro. Embryos at the 4-cell stage were produced by culturing embryos for 2 days after IVF and SCNT. After removal of the zona pellucida with acid Tyrode’s solution, three 4-cell-stage embryos (IVF3X) from IVF and two 4-cell-stage embryos (NT2X) from SCNT were aggregated by co-culturing them in an aggregation plate followed by culturing to the blastocyst stage. Embryos from IVF (IVF control) and SCNT (NT control) were also cultured to the blastocyst stage. All blastocysts were directly cultured on mitomycin C-inactivated murine embryonic fibroblasts as feeder layers. Two primary colonies were formed in the IVF control group (3.9%), whereas four primary colonies were formed in the IVF3X group (12.5%). One primary colony was formed in the NT2X group (20%), although no colony was formed in the NT control group. One of the IVF3X lines gradually disappeared after sub-passing, and the NT2X line also disappeared. Two ES-like cell lines derived from the IVF control were maintained up to 14 passages, and three ES-like lines from IVF3X were also maintained for more than 14 passages. These cells morphologically resembled human ES cells (flat and single layered) and expressed the markers of pluripotent cells such as alkaline phosphatase, NANOG, Oct-4, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. These results indicated that a porcine ES cell line could be established from in vitro-produced aggregated blastocysts. Further research is required to establish ES cell lines from SCNT embryos and characterize the differentiation and developmental abilities of these porcine ES-like cells. This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).

Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes

Development ◽  
1998 ◽  
Vol 125 (12) ◽  
pp. 2273-2282 ◽  
Author(s):  
W. Dean ◽  
L. Bowden ◽  
A. Aitchison ◽  
J. Klose ◽  
T. Moore ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Es Cells ◽  
Embryonic Stem ◽  
Upstream Region ◽  
Imprinted Genes ◽  
Epigenetic Alterations ◽  
Es Cell ◽  
Biallelic Expression

In vitro manipulation of preimplantation mammalian embryos can influence differentiation and growth at later stages of development. In the mouse, culture of embryonic stem (ES) cells affects their totipotency and may give rise to fetal abnormalities. To investigate whether this is associated with epigenetic alterations in imprinted genes, we analysed two maternally expressed genes (Igf2r, H19) and two paternally expressed genes (Igf2, U2af1-rs1) in ES cells and in completely ES cell-derived fetuses. Altered allelic methylation patterns were detected in all four genes, and these were consistently associated with allelic changes in gene expression. All the methylation changes that had arisen in the ES cells persisted on in vivo differentiation to fetal stages. Alterations included loss of methylation with biallelic expression of U2af1-rs1, maternal methylation and predominantly maternal expression of Igf2, and biallelic methylation and expression of Igf2r. In many of the ES fetuses, the levels of H19 expression were strongly reduced, and this biallelic repression was associated with biallellic methylation of the H19 upstream region. Surprisingly, biallelic H19 repression was not associated with equal levels of Igf2 expression from both parental chromosomes, but rather with a strong activation of the maternal Igf2 allele. ES fetuses derived from two of the four ES lines appeared developmentally compromised, with polyhydramnios, poor mandible development and interstitial bleeding and, in chimeric fetuses, the degree of chimerism correlated with increased fetal mass. Our study establishes a model for how early embryonic epigenetic alterations in imprinted genes persist to later developmental stages, and are associated with aberrant phenotypes.

Strategies for the isolation and characterization of bovine embryonic stem cells

1994 ◽  
Vol 6 (5) ◽  
pp. 569 ◽  
Author(s):  
RA Cherny ◽  
TM Stokes ◽  
J Merei ◽  
L Lom ◽  
MR Brandon ◽  
...  
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Farm Animals ◽  
Preimplantation Embryos ◽  
Es Cell ◽  
Isolation And Characterization

The practical application of advanced breeding technologies and genetic manipulation of domestic animals is dependent on the efficient and routine isolation of embryonic stem (ES) cell lines from these species. ES cell lines of proven totipotency have thus far been isolated only from the mouse. Murine ES cells can be identified by a number of criteria including morphology and characteristics in culture, the presence of specific markers, differentiative capacity and contribution to chimaeras. Reported cell lines derived from ruminant preimplantation embryos do not stably exhibit these characteristics. As demonstrated for the mouse, primordial germ cells may provide an alternative source for pluripotential cell lines. The isolation, culture and preliminary characterization of bovine primordial germ cell-derived (PGCd) cells are described in this paper. The PGCd cells are capable of differentiation in vitro and display murine ES cell markers including alkaline phosphatase. With farm animals, long generation intervals and small numbers of offspring make it important to develop techniques for evaluating chimaeric embryos in vitro before embarking on expensive in vivo programmes. A method for labelling putative pluripotential cells with a fluorochrome marker to follow the fate of such cells was developed. Labelled PGCd cells were injected into blastocysts and the chimaeric embryos were monitored in vitro. Preliminary results demonstrate that the labelled PGCd cells incorporate preferentially within the inner cell mass of the host blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS)

Embryonic Stem Cells: Prospects for Developmental Biology and Cell Therapy

2005 ◽  
Vol 85 (2) ◽  
pp. 635-678 ◽  
Author(s):  
Anna M. Wobus ◽  
Kenneth R. Boheler
Keyword(s):  
Developmental Biology ◽  
Cell Lines ◽  
Tissue Regeneration ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Populations ◽  
Es Cell ◽  
Mouse Es Cells ◽  
Human Es Cells

Stem cells represent natural units of embryonic development and tissue regeneration. Embryonic stem (ES) cells, in particular, possess a nearly unlimited self-renewal capacity and developmental potential to differentiate into virtually any cell type of an organism. Mouse ES cells, which are established as permanent cell lines from early embryos, can be regarded as a versatile biological system that has led to major advances in cell and developmental biology. Human ES cell lines, which have recently been derived, may additionally serve as an unlimited source of cells for regenerative medicine. Before therapeutic applications can be realized, important problems must be resolved. Ethical issues surround the derivation of human ES cells from in vitro fertilized blastocysts. Current techniques for directed differentiation into somatic cell populations remain inefficient and yield heterogeneous cell populations. Transplanted ES cell progeny may not function normally in organs, might retain tumorigenic potential, and could be rejected immunologically. The number of human ES cell lines available for research may also be insufficient to adequately determine their therapeutic potential. Recent molecular and cellular advances with mouse ES cells, however, portend the successful use of these cells in therapeutics. This review therefore focuses both on mouse and human ES cells with respect to in vitro propagation and differentiation as well as their use in basic cell and developmental biology and toxicology and presents prospects for human ES cells in tissue regeneration and transplantation.

scholarly journals Monkey Embryonic Stem Cell Lines Expressing Green Fluorescent Protein

2002 ◽  
Vol 11 (7) ◽  
pp. 631-635 ◽  
Author(s):  
Tatsuyuki Takada ◽  
Yutaka Suzuki ◽  
Yasushi Kondo ◽  
Nae Kadota ◽  
Kinji Kobayashi ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Transplantation ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Green Fluorescent

The major limitation of nonhuman primate (NHP) embryonic stem (ES) cell research is inefficient genetic modification and limited knowledge of differentiation mechanisms. A genetically modified NHP-ES cell with biomarkers, such as green fluorescent protein (GFP), that allow noninvasive monitoring of transgenic cells, is a useful tool to study cell differentiation control during preimplantation and fetal development, which also plays a crucial role in the development of cell transplantation medicine. Here we report the establishment of transgenic NHP-ES cell lines that express GFP without jeopardizing their pluripotency, which was confirmed by in vitro and in vivo differentiation. These GFP-expressing ES cells reproducibly differentiated into embryoid bodies, neural cells, and cardiac myocytes. They formed teratoma composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease (SCID) mice. GFP expression was maintained in these differentiated cells, suggesting that these cells were useful for cell transplantation experiments. Furthermore, we showed that these ES cells have the ability to form chimeric blastocysts by introducing into the early preimplantation stage NHP embryo.

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.

scholarly journals Mutant non-coding RNA resource in mouse embryonic stem cells

2021 ◽  
Vol 14 (2) ◽  
pp. dmm047803
Author(s):  
Jens Hansen ◽  
Harald von Melchner ◽  
Wolfgang Wurst
Keyword(s):  
Cell Lines ◽  
Large Scale ◽  
Es Cells ◽  
Single Gene ◽  
Embryonic Stem ◽  
Gene Trap ◽  
Es Cell ◽  
Lncrna Gene ◽  
Non Coding Rna

ABSTRACTGene trapping is a high-throughput approach that has been used to introduce insertional mutations into the genome of mouse embryonic stem (ES) cells. It is performed with generic gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA sequence tag for the rapid identification of the disrupted gene. Large-scale international efforts assembled a gene trap library of 566,554 ES cell lines with single gene trap integrations distributed throughout the genome. Here, we re-investigated this unique library and identified mutations in 2202 non-coding RNA (ncRNA) genes, in addition to mutations in 12,078 distinct protein-coding genes. Moreover, we found certain types of gene trap vectors preferentially integrating into genes expressing specific long non-coding RNA (lncRNA) biotypes. Together with all other gene-trapped ES cell lines, lncRNA gene-trapped ES cell lines are readily available for functional in vitro and in vivo studies.

scholarly journals 192ISOLATION AND CULTURE OF EMBRYONIC STEM CELL-LIKE CELLS FROM IN VITRO FERTILIZED PORCINE BLASTOCYSTS

2004 ◽  
Vol 16 (2) ◽  
pp. 217
Author(s):  
H.-Y. Son ◽  
C.-H. Park ◽  
S.-G. Lee ◽  
G.-S. Lee ◽  
H.-S. Kim ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Feeder Cells ◽  
Es Cell ◽  
Transgenic Pigs ◽  
Murine Embryonic Fibroblast

The establishment of porcine embryonic stem (ES) cell lines should be useful for the production of transgenic pigs and studies of developmental gene regulation. Recent development of techniques for production of embryos in vitro could be a useful source for the isolation of ES cells. Therefore, to establish porcine ES cells, this study was conducted to isolate and culture inner cell mass (ICM) from in vitro-fertilized (IVF) porcine blastocysts. Cumulus-oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Oocytes were then fertilized using a modified swim-up method to prevent polyspermy and cultured to the blastocyst stage. Initial culture of ICM was conducted after either culture of whole embryos or isolation of ICM by immunosurgery. Developing IVF embryos were continuously cultured in 50% DMEM and 50% F-10 with 15% fetal bovine serum, 1% non-essential amino acids, 1.7mM L-glutamine, 1% penicillin/streptomycin, 0.1mM α-mercaptoethanol, 1000 unit recombinant human LIF, 40ngmL−1 recombinant human SCF and 20ngmL−1 recombinant human basic FGF on a mytomycin-C-inactivated murine embryonic fibroblast (MEF) feeder layer. Antibodies against porcine cells were produced in rabbit. After removal of zona pellucida, ICMs were isolated by immunosurgery and cultured on feeder cells the same as described above. After IVF, the rates of 2-cell embryos and blastocysts were 70.8% and 20.4%, respectively. Results from the isolation and culture of ICMs of porcine blastocysts are shown in following table. ICM isolated by immunosurgery showed better attachment to feeder cells and ES cell colony formation than cultured whole blastocysts. Morphology of colonies was similar to that of mouse ES cells, showing compact colonies with delineated boundary. Also, these colonies showed alkaline phosphatase activity. Porcine ES-cell like colonies were passed 3 times through physical separation on fresh feeder layers. These results indicated that porcine ES-like cell line can be established from IVF porcine blastocysts. Further characterization of these porcine ES-like cell lines is required. Table 1 Isolation and culture of ICM from porcine blastocyst produced by IVF

Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

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