Blastocyst blastomeres

Human embryonic stem cells (hESCs) were first derived from the inner cell mass (ICM) from the blastocyst stage (-100-200 cells) of embryos generated by in vitro fertilization [35,36]. In humans the blastocyst is an early-stage embryo, approximately 4 to 5 days old. The blastocyst can be formed by means of either in vitro fertilization or somatic cell nuclear transfer, in which the nucleus of a somatic cell is combined with an enucleated oocyte. Methods have been developed to derive hESCs from the late morula stage (30-40 cells) and from arrested embryos (16-24 cells incapable of further development), and more recently from single blastomeres isolated from 8-cell embryos (37). 

The individual blastomeres of the mammalian embryo appear to be totipotent up to the 8-cell stage (Tarkowski, 1959 Tarkowski and Wroblewka, 1967 Moore et ah, 1968). By the blastocyst stage, two cell types have formed the outer trophoblast layer and the inner cell mass. These two populations have been shown to have restricted developmental capacities (Gardner, 1971). In mammalian embryos there is a characteristic grouping of the inner embryonic cell mass to one side... 

Although many offspring have been born, the efficiency of nuclear transplantation remains low. Two critical steps in the procedure have received most attention in the rabbit in particular (1) Activation of the oocyte has been improved by Collas and Robl (1990) with the use of multiple electrical stimulations. (2) Synchronization of donor blastomeres in Gi phase of the cell cycle prior to transplantation into enucleated metaphase II oocytes improved development of manipulated embryos to the blastocyst stage (Collas etal., 1992). Although synchronizing donor blastomeres in Gi improves development in vitro, the rate of development to term is still being determined. Therefore, procedures for synchronizing blastomeres in Gj phase are discussed here. This article presents protocols for the manufacture of micromanipulation pipettes, nuclear transplantation, electrically induced fusion and activation, and culture and transfer of reconstituted rabbit embryos. 

Berg, Biological Implications of electric field effects. V. Fusion of blastomeres and blastocysts of mouse embryos, Bloelectrochem. Bioenerg., 9 223-228 (1982). 

FIGURE 8.4 Alternate methods for deriving pluripotent stem cells. In addition to conventional derivation of ESCs from the ICM of blastocyst stage embryos, ESCs have also been derived from a single blastomere derived from eight-cell morulas, by injection of a somatic cell nucleus into an enucleated egg, or by treating somatic cells with defined factors for reprogramming the differentiation state. 

Most embryos used to generate blastomeres and blastocysts for hESC hne derivation are produced by in vitro fertilization. Somatic cell nuclear transfer (SCNT) provides an alternative approach to generating blastomeres and blastocysts. The nuclei of hESCs and adult primary fibroblasts have been transferred to unfertihzed human eggs to create blastocyst-stage embryos (French et al. 2008 Stojkovic et al. 2005). While this method has been used to establish animal ESC lines, it has not yet been demonstrated in humans. If the technical and ethical concerns surrounding therapeutic cloning can be addressed, nuclear transfer may allow the establishment of patient-specific hESC lines. 

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