scholarly journals Generation of mice derived from embryonic stem cells using blastocysts of different developmental ages

Reproduction ◽  
2008 ◽  
Vol 136 (5) ◽  
pp. 581-587 ◽  
Author(s):  
Hiroshi Ohta ◽  
Yuko Sakaide ◽  
Teruhiko Wakayama
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Embryonic Stem ◽  
Cell Number ◽  
The Other ◽  
Es Cell ◽  
Aggregation Method ◽  
Developmental Age

We previously showed that increasing the cell number of host tetraploid (4n) embryos by aggregating multiple 4n embryos at two to four-cell stages can improve the birthrate of mice from embryonic stem cells (ES mice). In the present study, we assessed whether in vitro aged blastocysts (e.g., E4.5 or E5.5), where their cell number also increased with development, can be used as hosts for generating ES mice. As expected, the cell number of in vitro aged 4n blastocysts increased with development, i.e., 26.5±2.4, 49.6±8.4, and 84.9±20.9 cells for E3.5, E4.5, and E5.5 respectively. Three independent ES cell lines were injected into 4n aged blastocysts, and their developmental ability was compared with that of E3.5 4n blastocysts commonly used for this procedure. We found that the birthrate of ES mice derived from E4.5 blastocysts were comparable with those of mice generated from E3.5 blastocysts. On the other hand, the birthrates decreased when E5.5 blastocysts were used. These results suggest that not only the cell number but also developmental age is important for producing ES mice. We also discuss a comparison of the present findings with those of our previous study, where ES mice were generated using an aggregation method employing the same ES cell lines.

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.

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.

scholarly journals Full-term development of enucleated mouse oocytes fused with embryonic stem cells from different cell lines

Reproduction ◽  
2001 ◽  
pp. 729-733 ◽  
Author(s):  
T Amano ◽  
Y Kato ◽  
Y Tsunoda
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Clear Correlation ◽  
Developmental Potential ◽  
Mouse Oocytes

The developmental potential of enucleated mouse oocytes receiving embryonic stem cells from ten lines with either the same or different genetic backgrounds using the cell fusion method was examined in vitro and in vivo. The development of nuclear-transferred oocytes into blastocysts was high (34-88%). However, there was no clear correlation between development into blastocysts after nuclear transfer and the chimaera formation rate of embryonic stem cells. The development into live young was low (1-3%) in all cell lines and 14 of 19 young died shortly after birth. Most of the live young had morphological abnormalities. Of the five remaining mice, two died at days 23 and 30 after birth, but the other three mice are still active at days 359 (mouse 1) and 338 (mice 4 and 5) after birth, with normal fertility. However, the reasons for the abnormalities and postnatal death of embryonic stem cell-derived mice are unknown.

Hematopoietic Differentiation of Human Embryonic Stem Cells in Vitro : Comparison of Three Cell Lines

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4787-4787
Author(s):  
Marion Brenot ◽  
Annelise Bennaceur-Griscelli ◽  
Marc Peschanski ◽  
Maria Teresa Mitjavila-Garcia
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Human Embryonic Stem Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem

Abstract Human embryonic stem cells (hES) isolated from the inner cell mass of a blastocyst have the ability to self renew indefinitely while maintaining their pluripotency to differentiate into multiple cell lineages. Therefore, hES represent an important source of cells for perspective cell therapies and serve as an essential tool for fundamental research, specifically for understanding pathophysiological mechanisms of human diseases for the development of novel pharmacological drugs. The generation of hematopoietic stem cells from hES may serve as an alternative source of cells for hematopoietic reconstitution following bone marrow transplantation and an interesting approach to understand early stages of hematopoietic development which are difficult to study in human embryos. Using two different methods, we have differentiated three hES cell lines (SA01, H1 and H9) into hematopoietic cells by generating embryoid bodies and co-culturing on the murine Op9 cell line. In both experimental approaches, we obtain cells expressing CD34 and when cultured in hematopoietic conditions, SA01 and H1 cell lines differentiate into various hematopoietic lineages as demonstrated by BFU-E, CFU-GM and CFU-GEMM colony formation, whereas H9 have almost exclusively granulo-macrophage differentiation. Cells composing these hematopoietic colonies express CD45, CD11b, CD31, CD41 and CD235 and staining with May Grundwald-Giemsa demonstrate neutrophil and erythrocyte morphology. These results demonstrate the capacity of hES to differentiate into mature hematopoietic cells in vitro. Nevertheless, there exist some quantitative and qualitative differences about hematopoietic differentiation between the hES cell lines used. However, we still have to evaluate their capacity to reconstitute hematopoiesis in vivo in an immune deficient mouse model. We will also be interested in developing in vitro methods to expand these hematopoietic precursor cells derived from hES which may be used as a viable source for future cell therapy.

Hematopoietic progenitor/stem cells immortalized byLhx2 generate functional hematopoietic cells in vivo

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 3939-3946 ◽  
Author(s):  
Perpétua Pinto do Ó ◽  
Karin Richter ◽  
Leif Carlsson
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Embryonic Stem ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Generate Functional ◽  
Hematopoietic Stem

Hematopoietic stem cells (HSCs) are unique in their capacity to maintain blood formation following transplantation into immunocompromised hosts. Expansion of HSCs in vitro is therefore important for many clinical applications but has met with limited success because the mechanisms regulating the self-renewal process are poorly defined. We have previously shown that expression of the LIM-homeobox gene Lhx2 in hematopoietic progenitor cells derived from embryonic stem cells differentiated in vitro generates immortalized multipotent hematopoietic progenitor cell lines. However, HSCs of early embryonic origin, including those derived from differentiated embryonic stem cells, are inefficient in engrafting adult recipients upon transplantation. To address whetherLhx2 can immortalize hematopoietic progenitor/stem cells that can engraft adult recipients, we expressed Lhx2 in hematopoietic progenitor/stem cells derived from adult bone marrow. This approach allowed for the generation of immortalized growth factor–dependent hematopoietic progenitor/stem cell lines that can generate erythroid, myeloid, and lymphoid cells upon transplantation into lethally irradiated mice. When transplanted into stem cell–deficient mice, these cell lines can generate a significant proportion of circulating erythrocytes in primary, secondary, and tertiary recipients for at least 18 months. Thus, Lhx2immortalizes multipotent hematopoietic progenitor/stem cells that can generate functional progeny following transplantation into lethally irradiated hosts and can long-term repopulate stem cell–deficient hosts.

scholarly journals 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.

The homeodomain protein Nanog and pluripotency in mouse embryonic stem cells

2005 ◽  
Vol 33 (6) ◽  
pp. 1518-1521 ◽  
Author(s):  
A. Yates ◽  
I. Chambers
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Leukaemia Inhibitory Factor ◽  
Homeodomain Protein ◽  
Es Cell ◽  
Homeodomain Proteins ◽  
Morphogenetic Protein

Intrinsic regulators of the pluripotency of mouse ES (embryonic stem) cells include the homeodomain proteins Oct4 and the recently identified Nanog. When overexpressed, Nanog displays the unique attribute of robustly sustaining ES cell self-renewal in the absence of the otherwise requisite extracellular stimulation by LIF (leukaemia inhibitory factor) and BMP (bone morphogenetic protein). Here, we review our current understanding of the function of Nanog in pluripotent stem cells both in vitro and in vivo.

Properties and uses of embryonic stem cells: prospects for application to human biology and gene therapy

1998 ◽  
Vol 10 (1) ◽  
pp. 31 ◽  
Author(s):  
P. D. Rathjen ◽  
J. Lake ◽  
L. M. Whyatt ◽  
M. D. Bettess ◽  
J. Rathjen
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Embryonic Stem ◽  
Cell Types ◽  
Human Biology ◽  
Pluripotent Cell ◽  
Cell Specification

Embryonic stem cells are pluripotent cells derived from the early mouse embryo that can be propagated stably in the undifferentiated state in vitro. They retain the ability to differentiate into all cell types found in an embryonic and adult mouse in vivo, and can be induced to differentiate into many cell types in vitro. Exploitation of ES cell technology for the creation of mice bearing predetermined genetic alterations has received widespread attention because of the sophistication that it brings to the study of gene function in mammals. Analysis of cell differentiation in vitro has also been of value, leading to the identification of novel bioactive factors and the elucidation of cell specification mechanisms. In this paper, we summarise the features of pluripotent cell lines and their applications, foreshadowing the impact that these systems may have on human biology. While the isolation of definitive human pluripotent cell lines has not yet been achieved, potential applications for these cells in the study of human biology, particularly cell specification, can be envisaged. Of particular interest is the possibility that human embryonic stem cells with properties similar to mouse embryonic stem cells might provide a generic system for gene therapy.

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