Variable Reprogramming of the Pluripotent Stem Cell Marker Oct4 in Mouse Clones: Distinct Developmental Potentials in Different Culture Environments

Abstract

A prevailing view of cloning by somatic-cell nuclear transfer is that reprogramming of gene expression occurs during the first few hours after injection of the nucleus into an oocyte, that the process is stochastic, and that the type of reprogramming needed for cloning success is foreign and unlikely to be readily achieved in the ooplasm. Here, we present evidence that the release of reprogramming capacity is contingent on the culture environment of the clone while the contribution of aneuploidy to altered gene expression is marginal. In particular, the rate of blastocyst formation in clones and the regional distribution of mRNA for the pluripotent stem cell marker Oct4 in clonal blastocysts was highly dependent on the culture environment after cumulus cell nuclear transfer, unlike that in genetically equivalent zygotes. Epigenetic modifications of genetically identical somatic nuclei continue after the first cell division of the clones and are amenable to a degree of experimental control, and their development to the blastocyst stage and appropriate expression of Oct4 predict further outcome, such as derivation of embryonic stem (ES) cells, but not fetal development. This observation indicates that development to the blastocyst stage is not equivalent to full reprogramming and lends support to the novel concept that ES cells are not the equivalent of the inner cell mass, hence the discrepancy between ES cell derivability and fetal development of clones.

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos

“…Considering that NT embryos are experimentally produced whereas fertilized embryos are made by the natural process, it is reasonable to think that transferred somatic nuclei undergo higher cellular stress than sperms. This notion is supported by the fact that NT embryos exhibit higher rates of apoptosis and weaker stress-coping functions than fertilized embryos (60)(61)(62). Nevertheless, the proportion of NT embryos that develop to the blastocyst stage is similar to that of in vitro fertilized embryos in many reports.…”

“…NT embryos undergo higher rates of apoptosis (60) and are less tolerant to the in vitro culture environment than fertilized embryos (61). Lower levels of gene expression related to cell protection from stress and apoptosis have been reported in NT embryos, compared with fertilized embryos (62).…”

Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1

“…Consequently, one major limiting step in somatic cell reprogramming, namely activation of the endogenous Oct4 locus (35,36), is overcome almost immediately after exogenous factor expression. In Oct4-GFP reporter MEFs, GFP + cells were detected as early as day 3 to 4 after expressing 6F, which is comparable to somatic cell nuclear transfer (37) or cell fusion (38). Therefore, technically, 6F should facilitate further development of nongenetic modifying reprogramming such as using episome and mRNA (39,40).…”

Reprogramming of Somatic Cell Identity

“…Although NT-ES cells and iPS cells have highly similar, if not identical, epigenetic and developmental potential characteristics, it is interesting to explore whether the mechanisms for inducing reprogramming by a defined factor or the oocyte cytoplasm are similar. An important observation from NT is that the key pluripotency gene Oct4 is reactivated in the somatic nucleus at the two to four cell stage in cloned embryos, suggesting that major reprogramming events can occur following one to two cell divisions (Boiani et al 2005). In contrast, direct in vitro reprogramming of different somatic cell types proceeds during a period of at least 3 weeks in mice and even longer in humans (Fig.…”