Embryo, mouse embryonic stem cells

Such cells are often classified on the basis of their original source as either embryonic or adult stem cells. As the name suggests, embryonic stem cells are derived from the early embryo, whereas adult stem cells are present in various tissues of the adult species. Much of the earlier work on embryonic stem cells was conducted using mouse embryos. Human embryonic stem cells were first isolated and cultured in the laboratory in 1998. Research on adult stem cells spans some four decades, with the discovery during the 1960s of haematopoietic stem cells in the bone marrow (Chapter 10). However, the exact distribution profile, role and ability to manipulate adult stem cells (particularly those outside of the bone marrow) are subjects of intense current research, and for which more questions remain than are answered. 

Osman AM et al (2010) Proteome profiling of mouse embryonic stem cells to define markers for cell differentiation and embryo-toxicity. Reprod Toxicol 30 322-332. doi S0890-6238(10)00193-0(pii)10.1016/j. reprotox.2010.05.084... 

In 2007, the DART committee held a workshop on alternative assays, which was followed up by a workshop held at the European Teratology Society Annual Meeting in 2009. These workshops focused on three alternative assays (1) whole embryo culture (WEC), (2) mouse embryonic stem cell tests (mESC), and (3) zebrafish. Each assay was presented and data from users were shared, and strengths and limitations were discussed. It should be noted that the WEC and mESC are validated by ECVAM as alternative embryotoxicity assays. Still, there are numerous research needs before even validated tests can achieve regulatory acceptance. The discussions, conclusions, and recommendations of the 2007 workshop were published by Chapin et al. (14). Bullet lists of next steps to move forward were defined for each assay (14) and are briefly summarized here ... 

Due to the limited applicability of in silico SAR approaches for developmental toxicity, there is more reliance on in vitro screening. From what has been publicly disclosed, it is evident that the four in vitro tests used for industrial screening are chick embryonic neural retina (CENR) micromass culture, whole embryo culture (WEC, rodent or rabbit), and mouse embryonic stem cells (EST). Recently, there has been significant interest within the pharmaceutical industry in the use of zebrafish for developmental toxicity testing,30 but because this aspect is in its infancy, there is little that has been publicly disclosed except limited abstracts and slide decks at several workshops.31 Although reviewed in considerable detail elsewhere,30-32 36 each test will be briefly compared and contrasted here. 

Figure 9-5 The sensitivity of the conceptus to a theoretical teratogen during rat gestation (modified from 161). The most susceptible window is organogenesis with low levels of vulnerability at the time of implantation and the period of functional maturation. Superimposed are the approximations of when the developmental landmarks that are represented in the four in vitro tests occur. The chick embryo neural retina model (CENR) represents events around GD 10-13. The mouse embryonic stem cell test (EST) corresponds roughly to the period of GD 6-10 in the rat, near the peak of sensitivity. Whole embryo culture (WEC) recapitulates the window at the peak of sensitivity, between GD 9-11 or GD 10-12 depending upon the window within which the culture is conducted. Rabbit cultures are also done between GD 10-12. Represented by the single ( ) and double asterisk ( ), respectively, are the initiation and termination of the dosing period in regulatory compliant preclinical embryo/fetal toxicity studies. Thus, the zebrafish is the only model that permits exposure to test article during this important period.

The EST has been developed with the aim to exploit the characteristics and differentiation potential of mouse embryonic stem cells (ES cells), established from the early embryo in 1981 [4, 5], ES cells are cultured in suspension to induce the formation of embryoid bodies, and afterwards they are transferred in 24-well dishes to allow attachment and differentiation in contracting cardiomyocytes. The toxicological endpoint is the inhibition of cardiac differentiation. In parallel a cytotoxicity test is performed on undifferentiated ES cells and a control somatic (fibroblast) cell line (3T3). The concentrations of testing chemicals that induce 50 % of differentiation inhibition (ID50) and 50 % cytotoxicity (IC50) in ES cells and 3T3 cells are inserted in a validated prediction model to classify the test chemical as non-embryotoxic, moderate, or strong embryotoxic [2, 6, 7], The validation of the method has been... 

ES-D3 Mouse embryo Pluripotent embryonic stem cell Insulin production... 

Suemori H, Kadodawa Y, Goto K, Araki I, Kondoh H, Nakatsuji N (1990) A mouse embryonic stem cell line showing pluripotency of differentiation in early embryos and ubiquitous beta-galactosidase expression. Cell Differ Dev 29 181-186. 

Additional studies have examined time-dependent molecular events associated with developmental toxicity in mouse embryos (102-104) and differentiating cardiomyocytes from embryonic stem cells (105). These initial time-response studies show the power and sensitivity of toxicogenomic assessments to characterize the timing of molecular changes that associate with developmental toxicity outcomes. 

Embryonic stem cells (ES cells) and homologous recombination are utilized to inactivate an endogenous gene from a host s genome. ES cell lines are derived from a 3-day embryo (ICM cells) and are undifferentiated but remain totipotent. Mouse... 

Beddington, R.S.P., Robertson, E.J. 1989. An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. Development 105, 733-737. 

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